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[Molecular diagnostics in urologic oncology. Detection of nucleic acids in urine samples].

The goal of molecular diagnostics in oncology is the early diagnosis of malignant disease processes during initial work-up or as part of follow-up. Body fluids serve as the primary material for non-invasive diagnostic methods. Besides actual tumor cells, the examination of urine can yield evidence of secreted proteins or even free nucleic acids. In principle, all of the methods available for the detection of tumor markers in tissue or blood samples can be successfully applied to the examination of urine samples. However, molecular biological examination of urine samples is associated with important problems because the cells in such samples are exposed to significant degradation and regression effects and because certain components of the urine act to inhibit the polymerase chain reaction. The present overview discusses the respective strengths and weakness of the available technology as applied to the diagnosis of urologic malignancies. Experimental studies conducted to date have reported high sensitivities and specificities for molecular diagnostics using urine samples. It is important to note that not only carcinomas of the urinary bladder can be diagnosed from material obtained in urine samples: in fact, the method can be used to diagnose entities such as renal cell and prostate carcinomas and, due to renal filtration of DNA, even non-urologic malignancies. The diagnostic application of these methods, however, remains in an experimental stage and must still clear several hurdles before becoming available for routine clinical use.

Biomarkers, Tumor↗

JAK2 V617F in myeloid disorders: molecular diagnostic techniques and their clinical utility: a paper from the 2005 William Beaumont Hospital Symposium on Molecular Pathology.

In early 2005, several groups of investigators studying myeloid malignancies described a novel somatic point mutation (V617F) in the conserved autoinhibitory pseudokinase domain of the Janus kinase 2 (JAK2) protein, which plays an important role in normal hematopoietic growth factor signaling. The V617F mutation is present in blood and marrow from a large proportion of patients with classic BCR/ABL-negative chronic myeloproliferative disorders and of a few patients with other clonal hematological diseases such as myelodysplastic syndrome, atypical myeloproliferative disorders, and acute myeloid leukemia. The JAK2 V617F mutation causes constitutive activation of the kinase, with deregulated intracellular signaling that mimics continuous hematopoietic growth factor stimulation. Within 7 months of the first electronic publication describing this new mutation, clinical molecular diagnostic laboratories in the United States and Europe began offering JAK2 mutation testing on a fee-for-service basis. Here, I review the various techniques used by research groups and clinical laboratories to detect the genetic mutation underlying JAK2 V617F, including fluorescent dye chemistry sequencing, allele-specific polymerase chain reaction (PCR), real-time PCR, DNA-melting curve analysis, pyrosequencing, and others. I also discuss diagnostic sensitivity, performance, and other practical concerns relevant to the clinical laboratorian in addition to the potential diagnostic utility of JAK2 mutation tests.

Base Sequence↗

Molecular diagnostics: a historical perspective.

The rapid growth in molecular diagnostic testing, which has averaged between 10% and 20% per year for the past 5 years, is largely attributable to both breakthroughs in our basic understanding (i.e., the Human Genome Project) and in applied technology. In the past decade, molecular applications have moved from labor-intensive and manual to rapid and automated due to improvements in sample extraction, target amplification, and sensitive and specific detection schema. This review describes some of the more significant technological milestones of the past 10 years and, when tied to basic and applied research, how these have led to important clinical applications. The next decade promises even more exciting technologies and applications for the field of molecular laboratory medicine.

Clinical Laboratory Techniques↗

Molecular diagnostic testing for infectious diseases using TMA technology.

Molecular diagnostic tests based on nucleic acid amplification technologies (NAATs) have become widely established in clinical microbiology laboratories in recent years. The acceptance of these tests has been driven by the development of more accurate and less labor-intensive commercial assay kits by diagnostic manufacturers. Infectious disease diagnostic assays using transcription-mediated amplification (TMA) NAAT have become increasingly popular in many clinical microbiology laboratories. Recent technology developments have improved the performance and simplified the use of the TMA assays. These new technologies have been applied to the development of multiplex TMA tests to improve the testing accuracy for organisms, such as Chlamydia trachomatis and Neisseria gonorrhoeae in clinical microbiology laboratories. TMA tests for HIV-1 and HCV have also led to improvements in blood bank testing which can improve the safety of the public blood supply.

Blood↗

Training costs and investment payback of implementing molecular diagnostics for identification of vancomycin resistant enterococci in a clinical microbiology laboratory.

Molecular diagnostics may be a more efficient method to manage resources; but most Microbiology laboratories have not introduced them into routine use due to the specialized training required. Using vancomycin resistant enterococci (VRE) screening during a comparison of a multiplex PCR (MPCR) and conventional biochemical testing (CBT) we studied 3 objectives: 1) to develop a molecular diagnostics in-house training program, 2) to assess the training program outcomes for competency and confidence, and 3) to determine laboratory payback. A training program for 14 technologists using multiple adult learning methods was implemented. Methods to minimize technical errors were introduced and included: use of a calibrated loop to deliver sample; prealiquotting reagents; increasing volume of specimen; addition of gel loading dye directly into reaction tubes; and establishment of an equivocal zone. In our laboratory MPCR costs $7.06 less than CBT, therefore the payback period for training and implementation would be approximately 3 years.

Cost-Benefit Analysis↗

Up close and personal: molecular diagnostics in oncology.

The almost overwhelming volume of information and new technological developments that has demanded so much of our scientific attention over the last decade will shortly revolutionize clinical diagnostics. Some of these developments are already affecting the working lives of scientists and clinicians alike, but will eventually require a greater understanding and acceptance from a much wider audience. Therefore it is important in our current scientific endeavor and commercial enthusiasm for molecular diagnostics that we maintain some awareness of the significant obstacles that must be overcome if we are to see an appropriate, timely and widespread adoption of molecular diagnostic testing in oncology. This article presents a brief commentary on the current state of the art in molecular diagnostics in oncology and how this relates to a more personalized approach to treatment.

Chromosome Mapping↗

Dynamic mutations pose unique challenges for the molecular diagnostics laboratory.

Routine clinical molecular testing of diseases associated with unstable or dynamic trinucleotide repeat syndromes poses unique technical, medical, and ethical challenges to the laboratory. Although the pathophysiology of these disorders is to date still largely undefined, the uniformity of their genetics has led to the development of highly informative diagnostic tests. In general, amplification techniques, such as the polymerase chain reaction (PCR), are used to determine the size of alleles within the genes linked to these disorders. Technically, these assays require empirical optimization so that the PCR reactions are both robust and reproducible, and occasionally other methods must be used to confirm diagnoses. Beyond execution of the test, however, the molecular diagnostics laboratory needs also to be fundamentally involved in the process of interpreting these tests in the correct clinical context and in setting policy as to how these data are presented to patients.

Chemistry, Clinical↗

Intramedullary Ewing sarcoma of the spinal cord: consequences of molecular diagnostics. Case report.

Molecular biological techniques have begun to transform modern medicine. These techniques have shown promise in the pathological diagnosis of difficult or uncommon tumors. Accurate molecular diagnosis of the small round-cell tumors, for example, is especially important because divergent therapies may be required to eradicate such disparate lesions as neuroblastoma, lymphoma, rhabdomyosarcoma, central primitive neuroectodermal tumors/medulloblastoma, or Ewing sarcoma (ES). The authors present an unusual case of a primary, extraosseous ES arising from the intramedullary spinal cord, in which molecular studies were required for specific diagnosis and therapeutic guidance.

Adult↗

Point-of-care molecular diagnostic systems--past, present and future.

The field of molecular diagnostics has greatly decreased the time it takes to identify infectious agents and to test their antimicrobial resistance. Portable devices are currently in development that can easily identify a variety of nucleic acid targets (either DNA or RNA) from multiple sample types in under an hour. This is done by a variety of methods including real-time polymerase chain reaction, probe-based assays, bioluminescence real-time amplification, and microarray or micro-pump technologies. These self-contained systems require only minimal training and perform all steps of the assay from extraction through detection with little or no operator intervention.

Humans↗

Aspects of molecular diagnostics and therapy in obstetrics and gynecology.

Scientific progress and information relating to the theoretical and clinical work being carried out in the field of obstetrics and gynecology has dramatically increased due to recent developments in molecular biology. Molecular obstetrics and gynecology is therefore the link between the different sections in obstetrics and gynecology. At present, the molecular understanding of cellular pathways is much greater than that of the direct integration of molecular diagnostics and therapy in routine clinical practice. The use of molecular diagnostics, such as preimplantation diagnostics or predictive genetic testing, still has technical problems as well as novel, and to date unclear, social, ethical and legal implications. To date, the technical elements of molecular therapy have not yet fulfilled their expectations. In the broad spectrum of obstetrics and gynecology, new molecular discoveries are influenced not only by technical but also by socioeconomic and political considerations. These include, for example, free access to genetic testing, patents for genes and the financial monopoly over molecular medication. Society must propose rules for the potential integration of the knowledge of molecular obstetrics and gynecology into the daily care of those seeking aid or advice.

Ethics, Medical↗

Molecular diagnostics for the detection of musculoskeletal infection.

The last decade has seen the crossover of basic molecular research techniques toward clinical application. The use of molecular diagnostics originally was driven by the desire to identify unculturable or fastidious pathogens. Success in this setting has led to new applications aimed at identifying organisms involved in musculoskeletal infection. Recently, polymerase chain reaction techniques have been investigated as to their efficacy in diagnosing septic arthritis and periprosthetic infection. This paper will offer an introduction to molecular diagnostics, a brief review of several techniques, and update their status in the diagnosis of musculoskeletal infection.

DNA, Bacterial↗

Multiplex RT-PCR for the detection of leukemia-associated translocations: validation and application to routine molecular diagnostic practice.

The aim of this study was to validate the application of a commercially available multiplex reverse transcription polymerase chain reaction (RT-PCR) assay [Hemavision-7 System] for the seven most common leukemia translocations for routine molecular diagnostic hematopathology practice. A total of 98 samples, comprising four groups, were evaluated: Group 1, 16 diagnostic samples molecularly positive by our existing laboratory-developed assays for PML-RARalpha/t (15;17) or BCR-ABL/t (9;22); Group 2, 51 diagnostic samples negative by our laboratory-developed assays for PML-RARalpha/t (15;17) or BCR-ABL/t (9;22); Group 3, 21 prospectively analyzed diagnostic cases, without prior molecular studies; and Group 4, 10 minimal residual disease (MRD) samples. Analysis of the two previously studied cohorts (Groups 1 and 2) confirmed the diagnostic sensitivity and specificity of the multiplex assay with regard to these two translocations. Additionally, however, in the "negative" Group (Group 2) the assay revealed three unanticipated translocations (CBFbeta-MYH11, BCR-ABL, and MLL-AF4), two of which were confirmed on cytogenetics. Analysis of the prospective cohort demonstrated that the assay was cost-effective and amenable to standard laboratory practice, with an identically sensitive MRD detection rate to that of our laboratory-developed tests. Virtually all of the results obtained were consistent with the phenotype and karyotype by conventional methods. This study illustrates the utility of a kit-based multiplex RT-PCR assay for the molecular diagnosis and monitoring of leukemias and reinforces the complementary roles of molecular testing and cytogenetics in diagnostic hematopathology.

Cell Line, Tumor↗

AgarCyto: a novel cell-processing method for multiple molecular diagnostic analyses of the uterine cervix.

In diagnostic cytology, it has been advocated that molecular techniques will improve cytopathological diagnosis and may predict clinical course. Ancillary molecular techniques, however, can be applied only if a sufficient number of preparations are made from a single cell sample. We have developed the AgarCyto cell block procedure for multiple molecular diagnostic analyses on a single scraping from the uterine cervix. The optimized protocol includes primary fixation and transport in ethanol/carbowax, secondary fixation in Unifix, and embedding in 2% agarose and then in paraffin according to a standard protocol for biopsies. More than 20 microscopic specimens were produced from a single AgarCyto cell block, and standard laboratory protocols have been successfully applied for H&E staining, immunohistochemistry for Ki-67 and p53, and in situ hybridization for the centromere of human chromosome 1 and human papilloma virus Type 16. In addition, single AgarCyto sections yielded sufficient input DNA for specific HPV detection and typing by LiPA-PCR, and the protocol includes an option for DNA image cytometry. The AgarCyto cell block protocol is an excellent tool for inventory studies of diagnostic and potentially prognostic molecular markers of cervical cancer.

Cell Line↗

Molecular diagnostic applications of DNA methylation technology.

Cancer arises due to the accumulation of DNA modifications that give cells a selective growth advantage. One common DNA modification is promoter hypermethylation associated with loss of expression of a tumor suppressor gene. The methylation status of a specific sequence or the pattern of methylation across the genome can be readily measured, and these sequences and analytical methods are being rapidly developed for molecular diagnostic applications. Detection of certain methylation events can be used for early detection of tumors, and analysis of patterns of methylation across the genome might provide information on disease subtype, aggressiveness, and treatment response. DNA methylation-based molecular diagnostic assays are particularly attractive because of the stability of the target analyte (DNA) and the potential sensitivity of the assays. As the field matures, methylation-based assays will make a major contribution to the field of molecular diagnostics, providing tools to fill unmet needs in current diagnostic and treatment plans for many types of cancer.

Biomarkers, Tumor↗

Molecular diagnostics: a healthcare perspective.

Recent progress in the development of molecular diagnostics in medicine has been rapid and the hope has been expressed by some people that it will soon be possible to have a detailed 'genetic readout' to assist in the diagnosis of treatment of a variety of diseases. However, such an outcome may be neither achievable nor desirable when viewed from the perspective of suppliers of healthcare.

Cost-Benefit Analysis↗

Molecular diagnostics of viral hemorrhagic fevers.

This review addresses the diagnostics of viral hemorrhagic fevers (VHFs). In the first part, an overview is given on classical methods of VHF diagnostics as well as novel molecular diagnostic tools. Currently available polymerase chain reaction (PCR) assays for diagnosis of VHF are summarized and discussed. In the second part, VHF diagnostics are described in particular for Lassa fever, yellow fever, and Crimean-Congo hemorrhagic fever, based on cases that were imported into or occurred within Europe. The third part is focussed on important differential diagnoses of VHF.

Adult↗

Cyclin A1 and gametogenesis in fertile and infertile patients: a potential new molecular diagnostic marker.

BACKGROUND: The study aim was to evaluate cyclin A1 mRNA expression levels as a potential molecular diagnostic parameter in the work-up of testicular tissue from fertile versus infertile patients. METHODS: Cyclin A1 expression was quantified in 55 cryopreserved testicular tissue specimens by fluorescence real-time RT-PCR. A conventional histological work-up was performed concomitantly in all tissue specimens with additional semi-thin sectioning in all cases of non-obstructive azoospermia (n = 12), maturation arrest (n = 17) and Sertoli cell-only syndrome (SCOS; n = 9). RESULTS: The mean (+/- SD) normalized cyclin A1 expression (N(CyclinA1)) was 3.82 +/- 2.23 relative gene expression (RGE) in tissue specimens with normal spermatogenesis, and 0.625 +/- 0.221 RGE in those with maturation arrest at the level of early spermatids. Only minimal N(CyclinA1) was detected in tissue specimens with spermatogonia only or maturation arrest at the level of primary spermatocytes (0.005 +/- 0.008). Cyclin A1 expression was absent in the majority of SCOS specimens (0.002 +/- 0.002). CONCLUSIONS: These investigations suggested that cyclin A1 expression is altered in cases of spermatogenic disorders. Moreover, the level of cyclin A1 mRNA expression correlates with gametogenic disorders and seems well suited for a molecular-diagnostic classification supplementing the histopathological evaluation of spermatogenic disorders.

Biomarkers↗