Showing papers in "Expert Review of Molecular Diagnostics in 2009"

Current advances in research and clinical applications of PLGA-based nanotechnology

Abstract: Co-polymer poly(lactic-co-glycolic acid) (PLGA) nanotechnology has been developed for many years and has been approved by the US FDA for the use of drug delivery, diagnostics and other applications of clinical and basic science research, including cardiovascular disease, cancer, vaccine and tissue engineering. This article presents the more recent successes of applying PLGA-based nanotechnologies and tools in these medicine-related applications. It focuses on the possible mechanisms, diagnosis and treatment effects of PLGA preparations and devices. This updated information will benefit to both new and established research scientists and clinical physicians who are interested in the development and application of PLGA nanotechnology as new therapeutic and diagnostic strategies for many diseases.

Gold nanoparticles for molecular diagnostics

Abstract: Gold nanoparticles (AuNPs) exhibit a unique phenomenon, known as surface plasmon resonance, which is responsible for their large absorption and scattering cross-sections, which are four to five orders of magnitude larger than those of conventional dyes. In addition, their optical properties can be controlled by varying their sizes, shapes and compositions. AuNPs can be easily synthesized and functionalized with different biomolecules including oligonucleotides. Numerous methods have been utilized for detecting AuNPs such as colorimetric, scanometric, fluorescence, surface-enhanced Raman scattering and electrochemical techniques. These unique aspects have permitted the development of novel AuNP-based assays for molecular diagnostics which promise increased sensitivity and specificity, multiplexing capability, and short turnaround times. AuNP-based colorimetric assays in particular show great potential in point-of-care testing assays. This review discusses properties of AuNPs and their utilization for the development of novel molecular assays.

MammaPrint™ 70-gene signature: another milestone in personalized medical care for breast cancer patients

Abstract: The MammaPrint assay (Agendia BV, The Netherlands) is the first fully commercialized microarray-based multigene assay designed to individualize treatment for patients with breast cancer. MammaPrint, the first assay to be cleared at the 510(k) level by the US FDA's new in vitro diagnostic multivariate index assay classification, is offered as a prognostic test for women under the age of 61 years with either estrogen receptor-positive or -negative, lymph node-negative breast cancer. Unlike the Oncotype DX assay (Genomic Health, CA, USA), this test requires freshly prepared tissues collected into an RNA preservative solution. The 70 genes that comprise the MammaPrint assay are focused primarily on proliferation with additional genes associated with invasion, metastasis, stromal integrity and angiogenesis. The Microarray In Node-negative Disease may Avoid Chemotherapy (MINDACT) trial, sponsored by the European Organization for Research and Treatment of Cancer, involves the assessment of patients in the adjuvant treatment setting by the standard clinicopathologic prognostic factors included on Adjuvant! Online and by the 70-gene MammaPrint assay. The following article will consider the basic biology, technology, ease of clinical use, level of clinical validation and potential clinical utility of this test.

Reliability of real-time reverse-transcription PCR in clinical diagnostics: gold standard or substandard?

Abstract: Molecular diagnostics is one of the major growth areas of modern medicine, with real-time PCR established as a qualitative and quantitative technology that is rapid, accurate and sensitive. The sequencing of the human genome, comprehensive genomic, mRNA and miRNA expression profiling of numerous cancer types, the ongoing identification of disease-associated polymorphisms and the expanding availability of genomic sequence information for human pathogens has opened the door to a wide range of translational applications for this technology. Consequently, novel real-time PCR assays have been developed for diagnosis and prognosis, treatment monitoring, transplant biology and pathogen detection, as well as more controversial uses such as lifestyle genotyping. However, this technology is still troubled by significant technical deficiencies. Hence its often-improper use as a clinical tool has important public health implications, most recently demonstrated through its association with the measles, mumps and rubella vaccine/autism controversy. This serves as a timely reminder of the indispensable requirement for careful experimental design, validation and analysis.

HE4 as a biomarker for ovarian and endometrial cancer management.

Abstract: Ovarian and endometrial cancer will be diagnosed in over 63,000 women in 2009, resulting in 22,000 deaths in the USA. Histologic screening, such as pap smears for detection of cervical cancer, is not feasible for these diseases given difficulty with access to the tissue. Thus, a serum- screening test using a biomarker or panel of biomarkers would be useful to aid in cancer diagnosis, detection of recurrence and as a means to monitor response to therapy. In this review, we focus on the human epididymis protein (HE)4 gene, which appears to have potential as a biomarker for both of these diseases. The structure and methods of detection of HE4 are discussed. Preliminary data show that HE4 may have more potential than cancer antigen 125 in discriminating benign from cancerous ovarian masses, and has the strongest correlation with endometrial cancer of all markers tested to date. Utilizing risk stratification, a panel of biomarkers including HE4 may ultimately be useful for detecting ovarian and endometrial cancer at an early stage in patients at high risk.

DNA methylation and cancer diagnosis: new methods and applications

Abstract: Methylation of cytosines in cytosine-guanine (CpG) dinucleotides is one of the most important epigenetic alterations in animals. The presence of methylcytosine in the promoter of specific genes has profound consequences on local chromatin structure and on the regulation of gene expression. Changes in DNA methylation play a central role in carcinogenesis. Hypermethylation and consecutive transcriptional silencing of tumor-suppressor genes has been documented in numerous cancers. The identification of target genes silenced by this modification has a great impact on diagnosis, classification, definition of risk groups and prognosis of cancer patients. Here we outline genome-wide techniques aiming at the identification of relevant methylated promoters. Methods and applications allowing clinicians to monitor the methylation of target genes will be also reviewed.

Autism genetics: emerging data from genome-wide copy-number and single nucleotide polymorphism scans

Abstract: Autism and related traits are highly heritable but cannot be explained by currently known genetic risk factors. Therefore, the advent of genome-wide single nucleotide polymorphism (SNP) and copy number variant (CNV) microarray technologies heralded identification of additional autism loci. CNVs associated with autism seem to show variable expressivity, also leading to other phenotypes, such as schizophrenia, mental retardation/developmental delay and epilepsy. Initial genome-wide SNP-association studies have each identified a single novel associated locus with modest effect. Based on the lessons from other complex common disease, larger sample sizes and meta-analyses are likely to identify additional SNP loci, and the genes implicated may act on multiple related disorders. Even if common alleles or rare variants hold little predictive value, neurodevelopmental pathways disrupted in autism may be identified. Future research might yet uncover common CNV risk loci and rare single nucleotide risk alleles, which are currently difficult to detect. The genetic architecture and phenotypic heterogeneity identified so far suggest additional approaches, such as population-based research and study of relevant neurobiological endophenotypes.

Assessing both genetic variation (SNPs/CNVs) and gene-environment interactions may lead to personalized gastric cancer prevention.

Abstract: Technological advances with next-generation DNA-sequencing and microarrays have revolutionized research on the human genome [1–3]. Three more genomes, including the first one for a cancer patient, have been entirely sequenced and published in Nature [4,5]. Could this rapid evolution in assessing human genetic variation be incorporated into cancer clinical practice [6–10] and enable a true overall personalized medicine [11]? Genetic variation describes the differences in both the coding and noncoding portions of our DNA and is what makes each of us unique. It can also contribute to our personalized susceptibility to disease. Exhaustive ana lysis of human SNPs has led to the identification of interesting SNP markers for certain disorders. Although less common than SNPs, copy number variations (CNVs) – gain or loss of segments of genomic DNA relative to a reference – have also been shown to be associated with several complex and common disorders such as cancer [12,13]. There has been an explosion in biomedical research for the identification of genetic variation-based personalized management of complex diseases. Over the last 2 years, genome-wide association studies (GWAS) have identified more than 100 new chromosomal regions at which more than 165 novel DNA variants influence risk of common human diseases and clinical phenotypes [2]. Given the poor prognosis of advanced gastric cancer, primary prevention represents a major research goal. If rational research could be translated successfully into targeted screening of the general population and individualized risk prediction-based preventive intervention, we could discuss for a revolution in the clinical management of gastric cancer. Potential highly effective, risk-guided prevention strategies could dramatically reduce incidence and mortality of the disease. In an era of rapid technological advance, based on the assessment of human genomic variation through whole-genome scans and personal genomics, how realistic is this expectation [1–3]?

Personalized cancer diagnostics and therapeutics.

Abstract: Dimitrios H Roukos studied Medicine at the University of Athens, Greece, and specialized in General Surgery at the JW Goethe-University Hospital in Frankfurt (1983–1989), Germany, where he received the title Dr med. Since 1990, Roukos has worked at Ioannina University School of Medicine. His current research activity includes the integration of both standard clinical and treatment data, and genetics/personal genomics/protein interactions/signaling pathways network data to predict a genotypic (QTLs) and phenotypic (cancer diversity) map. He also works to integrate systems biology, in silico, bioinformatics and reverse engineering data to achieve personalized cancer biomedicine. In the past he has published innovative models, algorithms, concepts and invited papers (including editorials and perspectives). He has also participated in keynote plenary presentations and lectures (Quest speaker international meetings). He has acheived over 1500 citations, a Hirsch factor of 28 and a total impact factor of over 600.

Th2 cells as targets for therapeutic intervention in allergic bronchial asthma

Abstract: Th2 cells play a central role in the pathogenesis of allergic bronchial asthma, since each of their characteristic cytokines such as IL-4, IL-5, IL-9 and IL-13 contributes to hallmarks of this disease, including airway eosinophilia, increased mucus production, production of allergen-specific IgE and development of airway hyper-responsiveness. Therefore, these cells are predisposed as target cells for therapeutic intervention. Experimental approaches targeted Th2-type effector cytokines, Th2-cell recruitment and Th2-cell development. Another strategy uses the immunomodulatory potential of tolerance-inducing cytokines such as IL-10 or of cytokines such as IL-12, IL-18 and IFN-gamma that are able to induce a counterbalancing Th1 immune response.

MMTV mouse models and the diagnostic values of MMTV-like sequences in human breast cancer

Abstract: Mouse mammary tumor virus (MMTV) long terminal repeat (LTR)-driven transgenic mice are excellent models for breast cancer as they allow for the targeted expression of various oncogenes and growth factors in neoplastic transformation of mammary glands. Numerous MMTV-LTR-driven transgenic mouse models of breast cancer have been created in the past three decades, including MMTV-neu/ErbB2, cyclin D1, cyclin E, Ras, Myc, int-1 and c-rel. These transgenic mice develop mammary tumors with different latency, histology and invasiveness, reflecting the oncogenic pathways activated by the transgene. Recently, homologous sequences of the env gene of MMTV have been identified in approximately 40% of human breast cancers, but not in normal breast or other types of cancers, suggesting possible involvement of mammary tumor virus in human breast carcinogenesis. Accumulating evidence demonstrates the association of MMTV provirus with progesterone receptor, p53 mutations and advanced-stage breast cancer. Thus, the detection of MMTV-like sequences may have diagnostic value to predict the clinical outcome of breast cancer patients.

Mea Culpa with cancer-targeted therapy: new thinking and new agents design for novel, causal networks-based, personalized biomedicine.

Abstract: Over the last 10 years, there has been an explosion of interest in targeted agents for the treatment of cancer. Despite the standardization of surgery, radiotherapy and empirical chemotherapy, mill...

mTOR in renal cell cancer: modulator of tumor biology and therapeutic target.

Abstract: Elucidation of the crucial role of the PI3K/Akt/mTOR pathway in the pathogenesis of cancer has led to the development of various drugs targeting this signaling cascade at distinct levels. mTOR, a serine/threonine kinase plays a pivotal role in coupling growth stimuli to cell cycle progression. There are two distinct macromolecular complexes of mTOR: mTORC1, which is rapamycin-sensitive and contains raptor; and mTORC2, which is rapamycin-insensitive and contains rictor. However, in recent preclinical studies a sustained exposure of cancer cells to rapamycin has been shown to inhibit the function of both mTORC1 and mTORC2 complexes. Downstream targets of these complexes, which involve HIF-1alpha and HIF-2alpha, cyclin D1 and PKC-alpha, are responsible for the activation of various intracellular processes leading to the activation of cell proliferation, and induction of angiogenesis, metastasis or chemoresistance. Since the biology of renal cell cancer (RCC) is tightly controlled by mTOR, targeted inhibition of mTOR function appeared to be a promising therapeutic approach for RCC patients. To date, results of two, large, Phase III clinical trials evaluating the efficacy of rapamycin derivatives (i.e., temsirolimus and everolimus) in the treatment of RCC have been published. First-line temsirolimus (CCI-779) administered to metastatic, poor-prognosis RCC patients significantly prolonged overall and progression-free survival when compared with IFN-alpha. Treatment of metastatic RCC patients refractory to tyrosine kinase inhibitors with everolimus (RAD-001) significantly prolonged progression-free survival when compared with placebo. Therapeutic strategies based on mTOR inhibition in RCC demonstrated a significant clinical activity. However, there are still patients refractory to mTOR inhibitors. Various molecular mechanisms of resistance to rapalogues have been identified and will have to be targeted simultaneously with mTOR in order to achieve a complete inhibition of signaling pathways crucial for the pathogenesis of RCC. Such clinical trials evaluating a combination of mTOR inhibitors with other targeted therapies are ongoing.

DNA methylation changes in prostate cancer: current developments and future clinical implementation.

Abstract: Promoter hypermethylation is associated with the loss of expression of tumor-suppressor genes in cancer. Currently, several genome-wide technologies are available and have been utilized to examine the extent of DNA methylation in discovery-based studies involving several physiological and disease states. Although early in the process, aberrant DNA methylation is gaining strength in the fields of cancer risk assessment, diagnosis and therapy monitoring in different cancer types. There is a need to improve existing methods for early diagnosis of prostate cancer and to identify men at risk for developing aggressive disease. Because of the ubiquity of DNA methylation changes and the ability to detect methylated DNA in several body fluids (e.g., blood and urine), this specifically altered DNA may serve, on one hand, as a possible new screening marker for prostate cancer and, on the other hand, as a tool for therapy monitoring in patients having had neoplastic disease of the prostate. Since many prostate cancer patients present with advanced disease and some present with nonspecific elevation of prostate-specific antigen without prostate cancer, early detection with high specificity and sensitivity is considered to be one of the most important approaches to reduce mortality and unwanted tension of the men with high prostate-specific antigen. Therefore, an effective screening test would have substantial clinical benefits. Furthermore, methylation markers of risk of progression of disease in patients having prostate cancer permits immediate commencement of specific treatment regimens and probably longer survival and better quality of life. This review illustrates the current benefits and limitations of potentially useful prostate cancer methylation markers that have considerable existing data and touches upon other future markers as well as the field of methylation in prostate cancer.

Placentation abnormalities in the pathophysiology of preeclampsia.

Abstract: Preeclampsia, a pregnancy-specific syndrome of hypertension and proteinuria, is one of the leading causes of perinatal morbidity and mortality. Although the initiating factor is unknown, a clearer picture regarding the network mechanisms of preeclampsia has begun to emerge during the past few years. The pathogenesis of preeclampsia can be described as having two stages. The first stage of preeclampsia involves abnormal placental implantation, followed by transition to the second stage of endothelial dysfunction. The link bridging these two stages is considered to be oxidative stress and disrupting angiogenesis. Discovery of the fundamental molecular mechanisms causing preeclampsia may provide a potential for prophylactic intervention and symptom amelioration. In this article, the clinical syndrome and risk factors of preeclampsia will be introduced, followed by the recent advances in our understanding of the pathophysiology of preeclampsia and, finally, research into aberrant placentation will be discussed.

Current molecular diagnostics of breast cancer and the potential incorporation of microRNA.

Abstract: Although comprehensive molecular diagnostics and personalized medicine have sparked excitement among researchers and clinicians, they have yet to be fully incorporated into today's standard of care. This is despite the discovery of disease-related oncogenes, tumor-suppressor genes and protein biomarkers, as well as other biological anomalies related to cancer. Each year, new tests are released that could potentially supplement or surpass standard methods of diagnosis, including serum, protein and gene expression analyses. All of these novel approaches have shown great promise, but initial enthusiasm has diminished as difficulties in reproducibility, expense, standardization and proof of significance beyond current protocols have emerged. This review will focus on current and novel molecular diagnostic tools applied to breast cancer with special attention to the exciting new field of microRNA analysis.

Handling small supernumerary marker chromosomes in prenatal diagnostics.

Abstract: Small supernumerary marker chromosomes (sSMCs) are structurally abnormal chromosomes that cannot be thoroughly characterized by conventional banding cytogenetics and are equal in size or smaller than chromosome 20. They are present in 0.075% of prenatal cases and, overall, approximately 3 million people worldwide are carriers of a sSMC. In prenatal cases with ultrasound abnormalities, sSMCs are found in up to approximately 0.2% of the cases. First described in 1961, it is now known that sSMCs have no phenotypic effects in approximately 70% of de novo cases. Nonetheless, in at least 30-50% of prenatally detected sSMC cases, the pregnancy is terminated; that is, for a certain percentage of potentially healthy children with a sSMC, an abortion is induced. This situation can only be improved by providing increased amounts of and more reliable information on sSMCs. This article provides an overview on current state-of-the-art technologies and how sSMC analysis can be optimized in prenatal diagnostics.

Microelectrical sensors as emerging platforms for protein biomarker detection in point-of-care diagnostics.

Abstract: Current methods used to measure protein expression on microarrays, such as labeled fluorescent imaging, are not well suited for real-time, diagnostic measurements at the point of care. Studies have shown that microelectrical sensors utilizing silica nanowire, impedimetric, surface acoustic wave, magnetic nanoparticle and microantenna technologies have the potential to impact disease diagnosis by offering sensing characteristics that rival conventional sensing techniques. Their ability to transduce protein binding events into electrical signals may prove essential for the development of next-generation point-of-care devices for molecular diagnostics, where they could be easily integrated with microarray, microfluidic and telemetry technologies. However, common limitations associated with the microelectrical sensors, including problems with sensor fabrication and sensitivity, must first be resolved. This review describes governing technical concepts and provides examples demonstrating the use of various microelectrical sensors in the diagnosis of disease via protein biomarkers.

Helicase-dependent amplification: use in OnChip amplification and potential for point-of-care diagnostics

Abstract: Isothermal amplification technologies are emerging on the horizon that could have the potential to pose as alternatives to PCR in terms of sensitivity and ease of use. One of the most recent isothermal technologies is helicase-dependent amplification (HDA). This technology uses the helicase's capability to disrupt the hydrogen bonds of a Watson-Crick base pair in order to separate dsDNA. A denaturation step, as is used in PCR, is no longer required. This gives rise to new, less expensive and less complicated designs for point-of-care devices and 'Lab on Chip' systems. Helicase-dependent OnChip-amplification (OnChip-HDA) is a further step into this direction as it integrates the HDA technology with microarray technology and its power of multiplexing. This special report will give an overview on the HDA and OnChip-HDA technology, and its potential for point-of-care diagnostics.

Pharmacogenomics and therapeutic strategies for dementia.

Abstract: Recent advances in genomic medicine have contributed to the acceleration of our understanding regarding the pathogenesis of dementia, improving diagnostic accuracy with the introduction of novel biomarkers and personalizing therapeutics with the incorporation of pharmacogenetic and pharmacogenomic procedures to drug development and clinical practice. Most neurodegenerative disorders, including Alzheimer's disease (AD), share some common features, such as a genomic background in which hundreds of genes might be involved, genome-environment interactions, complex pathogenic pathways, poor therapeutic outcomes and chronic disability. The main aim of a cost-effective treatment is to halt disease progression via modification of the functional cascade involving AD genomics, transcriptomics, proteomics and metabolomics. Unfortunately, the drugs available for the treatment of dementia are not cost effective. The pharmacological treatment of dementia accounts for 10-20% of direct costs, and fewer than 20% of the patients are moderate responders to conventional drugs, some of which may cause important adverse drug reactions. Future antidementia drugs must address the complex pathogenic niche of the disease from a multifactorial perspective. Pharmacogenetic and pharmacogenomic factors may account for 60-90% of drug variability in drug disposition and pharmacodynamics. In addition to antidementia drugs, patients with AD or with other forms of dementia need concomitant medications for the treatment of diverse disorders of the CNS associated with progressive brain dysfunction. Approximately 60-80% of drugs acting on the CNS are metabolized via enzymes of the CYP gene superfamily, and 10-20% of Caucasians are carriers of defective CYP2D6 polymorphic variants that alter the metabolism of many psychotropic agents. Only 26% of the patients are pure extensive metabolizers for the trigenic cluster integrated by allelic variants of the CYP2D6, CYP2C19 and CYP2C9 in combination. Although many genes have been suggested to be associated with AD, with the exception of APOE, most polymorphic variants of potential risk exhibit a very weak association with AD. APOE-4/4 carriers exhibit a dramatic biological disadvantage in comparison with other genotypes, and AD patients harboring this homozygous condition are the worst responders to conventional drugs. The incorporation of pharmacogenetic/pharmacogenomic protocols into AD research and clinical practice can foster the optimization of therapeutics by helping to develop cost-effective biopharmaceuticals and improving drug efficacy and safety.

Serum proteomics of glioma: methods and applications.

Abstract: The prognosis of patients with glioblastoma, the most malignant adult glial brain tumor, remains poor in spite of advances in treatment procedures, including surgical resection, irradiation and chemotherapy. Genetic heterogeneity of glioblastoma warrants extensive studies in order to gain a thorough understanding of the biology of this tumor. While there have been several studies of global transcript profiling of glioma with the identification of gene signatures for diagnosis and disease management, translation into clinics is yet to happen. Serum biomarkers have the potential to revolutionize the process of cancer diagnosis, grading, prognostication and treatment response monitoring. Besides having the advantage that serum can be obtained through a less invasive procedure, it contains molecules at an extraordinary dynamic range of ten orders of magnitude in terms of their concentrations. While the conventional methods, such as 2DE, have been in use for many years, the ability to identify the proteins through mass spectrometry techniques such as MALDI-TOF led to an explosion of interest in proteomics. Relatively new high-throughput proteomics methods such as SELDI-TOF and protein microarrays are expected to hasten the process of serum biomarker discovery. This review will highlight the recent advances in the proteomics platform in discovering serum biomarkers and the current status of glioma serum markers. We aim to provide the principles and potential of the latest proteomic approaches and their applications in the biomarker discovery process. Besides providing a comprehensive list of available serum biomarkers of glioma, we will also propose how these markers will revolutionize the clinical management of glioma patients.

Influenza genome analysis using pyrosequencing method: current applications for a moving target.

Abstract: Pyrosequencing is a high-throughput non-gel-based DNA sequencing method that was introduced in the late 1990s. It employs a DNA sequencing-by-synthesis approach based on real-time measurement of pyrophosphate released from incorporation of dNTPs. A cascade of enzymatic reactions proportionally converts the pyrophosphate to a light signal recorded in a form of peaks, known as pyrograms. Routinely, a 45-60-nucleotide sequence is obtained per reaction. Recent improvements introduced in the assay chemistry have extended the read to approximately 100 nucleotides. Since its advent, pyrosequencing has been applied in the fields of microbiology, molecular biology and pharmacogenomics. The pyrosequencing approach was first applied to analysis of influenza genome in 2005, when it played a critical role in the timely detection of an unprecedented rise in resistance to the adamantane class of anti-influenza drugs. More recently, pyrosequencing was successfully applied for monitoring the emergence and spread of influenza A (H1N1) virus resistance to oseltamivir, a newer anti-influenza drug. The present report summarizes known applications of the pyrosequencing approach for influenza genome analysis with an emphasis on drug-resistance detection.

Human chorionic gonadotropin and associated molecules.

Abstract: When considering human chorionic gonadotropin (hCG) and hCG tests, it is important to realize that it is not a single biological molecule. The regular form of hCG produced by differentiated syncytotrophoblast cells (regular hCG) is a hormone made with the primary function of maintaining the myometrial and decidual spiral arteries and the vascular supply of the placenta during the full course of pregnancy. Hyperglycosylated hCG (hCG with double-size O-linked oligosaccharides) is made by undifferentiated cytotrophoblast cells. This is an autocrine hormone with separate functions, it maintains invasion as in implantation of pregnancy and malignancy in gestational trophoblastic diseases. A hyperglycosylated free β-subunit is produced by a high proportion of all malignancies. This functions as an autocrine hormone to promote the growth and invasion of the malignancy. It is important to realize when ordering an hCG test what you are measuring and whether the test ordered will detect appropriately these three va...

Mechanisms of resistance to ionizing radiation in rectal cancer.

Abstract: While patients with breast cancers are not subjected to the adverse side effects of tamoxifen or trastuzumab if their tumors are negative for estrogen, progesterone or Her-2/Neu, neoadjuvant ionizing radiation with concurrent chemotherapeutic agents is administered almost universally to patients with stage II/III rectal cancers. There is, however, a tremendously wide range of response to this preoperative modality from complete pathological response to continuous tumor growth in patients receiving the same form of treatment. The specific phenotype of the tumor plays a major role in rendering tumor cells survival advantage to the cytotoxic effects of chemoradiation. Pathways such as proliferation, cell cycle, apoptosis and hypoxia have been investigated under a variety of conditions in preirradiated tissues and postirradiated tumors. This article reviews the current evidence available to identify a molecular profile predictive of the best response to ionizing radiation.

Tools for stools: the challenge of assessing human intestinal microbiota using molecular diagnostics

Abstract: The human GI tract is inhabited by an incredibly complex and abundant microbiota, whose composition is dependent on a variety of factors. The gut microbiota has an influence in the morphological, immunological and nutritional functions of the digestive tract and may be involved in many diseases. This article proposes the rationale behind conducting in vitro diagnostics (IVDs) of the human microbiota, as well as outlining the conceptual and technical difficulties involved in IVD testing. The molecular methods that can be used according to whether the IVD tools are employed to study one individual constituent species or to determine the microbiota as a whole will also be described. In the latter case, these technologies include high-throughput sequencing for metagenomics and DNA microarrays, which can now be efficiently used to study gut ecology and are believed to represent the future of standardized diagnostics.

Epigenetic biomarkers in urothelial bladder cancer.

Abstract: Bladder cancers comprise heterogeneous cell populations, and numerous factors are likely to be involved in dictating recurrence, progression and patient survival. While several molecular markers that are used to evaluate the development and prognosis of bladder cancer have been studied, the limited value of these established markers has created the need for new molecular indicators of bladder cancer prognosis. Of particular interest is the silencing of tumor-suppressor genes by epigenetic alteration. Recent progress in understanding epigenetic modification and gene silencing has led to new opportunities for the understanding, detection, treatment and prevention of cancer. Moreover, epigenetic silencing of tumor-suppressor genes is interesting from a clinical standpoint, because of the possibility of reversing epigenetic changes and restoring gene function in a cell. This review focuses on the prognostic relevance of epigenetic markers in bladder cancer.

Advances in viral disease diagnostic and molecular epidemiological technologies

Abstract: The early and rapid detection and characterization of specific nucleic acids of medico-veterinary pathogens have proven invaluable for diagnostic purposes. The integration of amplification and signal detection systems, including online real-time devices, have increased speed and sensitivity and greatly facilitated the quantification of target nucleic acids. They have also allowed for sequence characterization using melting or hybridization curves. The newer-generation molecular diagnostic technologies offer, hitherto, unparalleled detection and discrimination methodologies, which are vital for the positive detection and identification of pathogenic agents, as well as the effects of the pathogens on the production of antibodies. The development phase of the novel technologies entails a thorough understanding of accurate diagnosis and discrimination of present and emerging diseases. The development of novel technologies can only be successful if they are transferred and used in the field with a sustainable quality-assured application to allow for the optimal detection and effective control of diseases. The aim of these new tools is to detect the presence of a pathogen agent before the onset of disease. This manuscript focuses mainly on the experiences of two World Organisation for Animal Health collaborating centers in context to molecular diagnosis and molecular epidemiology of transboundary and endemic animal diseases of viral origin, food safety and zoonoses.

Integrating genomics, proteomics and bioinformatics in translational studies of molecular medicine.

Abstract: Understanding the molecular mechanisms of disease requires the introduction of molecular diagnostics into medical practice. Current medicine employs only elements of molecular diagnostics, which are usually applied on the scale of single genes. Medicine in the postgenomic era will utilize thousands of disease-associated molecular markers provided by high-throughput sequencing and functional genomic, proteomic and metabolomic studies. Such a spectrum of techniques will link clinical medicine based on molecularly oriented diagnostics with the prediction and prevention of disease. To achieve this task, large-scale and genome-wide biological and medical data must be combined with biostatistical and bioinformatic analyses to model biological systems. Collecting, cataloging and comparing data from molecular studies, and the subsequent development of conclusions, creates the fundamentals of systems biology. This highly complex analytical process reflects a new scientific paradigm known as integrative genomics.

Human chorionic gonadotropin tests

Abstract: Human chorionic gonadotropin (hCG) is not a single biological molecule. There is the regular form of hCG produced by differentiated syncytotrophoblast cells (regular hCG). This hormone primarily functions to maintain the myometrial and decidual spiral arteries, or the vascular supply of the placenta during the full course of pregnancy. Hyperglycosylated hCG is made by undifferentiated cytotrophoblast cells, which are extravillous cytotrophoblast cells. This is an autocrine with separate functions, it maintains trophoblast invasion as in implantation of pregnancy and malignancy in gestational trophoblastic diseases. A hyperglycosylated free beta-subunit is produced by a high proportion of all malignancies. This also functions as an autocrine by promoting the growth and invasion of the malignancy. When ordering an hCG test it is important to realize what is being measured and whether the test ordered will detect appropriately these three variants of hCG as well as their degradation products. Most automated commercial laboratory tests, point-of-care tests and over-the-counter tests are limited in what they detect, focusing only on regular hCG. This is in part due to the US FDA, who only consider regular hCG as part of a pregnancy test, and to whom only detection of regular hCG is necessary. This may cause test errors since primarily hyperglycosylated hCG is produced in early pregnancy and in choriocarcinoma and germ cell testicular malignancies. Only free beta-subunits may be produced in other germ cell malignancies (all applications for hCG test). The exceptions are the older style hCGb radioimmunoassay and the Siemens Immulite platform hCG test, which detect all beta-subunit variants of hCG and their degradation product appropriately. Apart from test specificity limitations, assays for hCG and its variants are widely used clinically in pregnancy detection, early pregnancy detection, prediction of spontaneously aborting, and ectopic pregnancies and prediction of trisomy pregnancies. hCG tests are essential in managing gestational trophoblastic diseases, whether hydatidiform mole, invasive mole or choriocarcinoma, and are very useful in management of testicular malignancies and other germ cell malignancies.

Emergence of single-molecule sequencing and potential for molecular diagnostic applications.

Abstract: The effective demonstration of single-molecule sequencing at scale over the last several years offers the exciting opportunity for a new era in the field of molecular diagnostics. As we aim to personalize and deliver cost-effective healthcare, we must consider the need to fully integrate genomics into decision-making. We must be able to accurately and cost effectively obtain a complete genome sequence for disease diagnosis, interrogate a molecular signature from blood for therapeutic monitoring, obtain a tumor mutation profile for optimizing therapeutic choice - each molecular diagnostic measurement utilized to better inform patient care. Would a physician or molecular pathology laboratory want to utilize a PCR process in which millions of DNA copies of a patient's nucleic acid are created when an alternative approach allowing direct measurement of the nucleic acids is possible? I would suggest not! In this article we will focus on the emergence of single-molecule sequencing, the single-molecule sequencing methodologies in the marketplace or under development today, as well as the importance of these methods for molecular characterization and diagnosis of disease with the ultimate application for molecular diagnostics.