Early Detection of Cancer: Evaluation of MR Imaging Grading Systems in Patients with Suspected Nasopharyngeal Carcinoma.

BACKGROUND AND PURPOSE: We evaluated modifications to our contrast-enhanced MR imaging grading system for symptomatic patients with suspected nasopharyngeal carcinoma, aimed at improving discrimination of early-stage cancer and benign hyperplasia. We evaluated a second non-contrast-enhanced MR imaging grading system for asymptomatic patients from nasopharyngeal carcinoma plasma screening programs. MATERIALS AND METHODS: Dedicated nasopharyngeal MR imaging before (plain scan system) and after intravenous contrast administration (current and modified systems) was reviewed in patients from a nasopharyngeal carcinoma-endemic region, comprising 383 patients with suspected disease without nasopharyngeal carcinoma and 383 patients with nasopharyngeal carcinoma. The modified and plain scan systems refined primary tumor criteria, added a nodal assessment, and expanded the system from 4 to 5 grades. The overall combined sensitivity and specificity of the 3 systems were compared using the extended McNemar test (a χ2 value [Formula: see text]> 5.99 indicates significance). RESULTS: The current, modified, and plain scan MR imaging systems yielded sensitivities of 99.74%, 97.91%, and 97.65%, respectively, and specificities of 63.45%, 89.56% and 86.42%, respectively. The modified system yielded significantly better performance than the current ([Formula: see text] = 122) and plain scan ([Formula: see text] = 6.1) systems. The percentages of patients with nasopharyngeal carcinoma in grades 1-2, grade 3, and grades 4-5 for the modified and plain scan MR imaging systems were 0.42% and 0.44%; 6.31% and 6.96%; and 90.36% and 87.79%, respectively. No additional cancers were detected after contrast administration in cases of a plain scan graded 1-2. CONCLUSIONS: We propose a modified MR imaging grading system that improves diagnostic performance for nasopharyngeal carcinoma detection. Contrast was not valuable for low MR imaging grades, and the plain scan shows potential for use in screening programs.

Complementary roles of MRI and endoscopic examination in the early detection of nasopharyngeal carcinoma.

BACKGROUND: Early-stage nasopharyngeal carcinoma (NPC) evades detection when the primary tumor is hidden from view on endoscopic examination. Therefore, in a prospective study of subjects being screened for NPC using plasma Epstein-Barr virus (EBV) DNA, we conducted a study to investigate whether magnetic resonance imaging (MRI) could detect endoscopically occult NPC. PATIENTS AND METHODS: Participants with persistently positive EBV DNA underwent endoscopic examination and biopsy when suspicious for NPC, followed by MRI blinded to the endoscopic findings. Participants with a negative endoscopic examination and positive MRI were recalled for biopsy or surveillance. Diagnostic performance was assessed by calculating sensitivity, specificity and accuracy, based on the histologic confirmation of NPC in the initial study or in a follow-up period of at least two years. RESULTS: Endoscopic examination and MRI were performed on 275 participants, 34 had NPC, 2 had other cancers and 239 without cancer were followed-up for a median of 36 months (24-60 months). Sensitivity, specificity and accuracy were 76.5%, 97.5% and 94.9%, respectively, for endoscopic examination and 91.2%, 97.5% and 96.7%, respectively, for MRI. NPC was detected only by endoscopic examination in 1/34 (2.9%) participants (a participant with stage I disease), and only by MRI in 6/34 (17.6%) participants (stage I = 4, II = 1, III = 1), two of whom had stage I disease and follow-up showing slow growth on MRI but no change on endoscopic examination for 36 months. CONCLUSION: MRI has a complementary role to play in NPC detection and can enable the earlier detection of endoscopically occult NPC.

Detection of circulating fetal nucleic acids: a review of methods and applications.

The discovery of cell-free circulating fetal nucleic acids in maternal plasma has opened up new possibilities in non-invasive prenatal diagnosis. The rapid advancement of this field in the past decade is catalysed by the discovery of new classes of fetal nucleic acid markers and technological developments in nucleic acid detection and amplification. In this review, some of the more significant recent developments in this field will be discussed, including the detection of single molecule, chromosomal aneuploidies, single nucleotide variations and placental microRNAs in maternal plasma.

Circulating nucleic acid analysis: diagnostic applications for acute pathologies.

Much research interest has been shown in recent years for the development of molecular diagnostic strategies based on the analysis of DNA/RNA molecules that are present in the plasma/serum of human subjects. Reported applications include the diagnosis, prognostication or monitoring of malignancies and pregnancy-associated complications. While researchers have speculated that cell death is a potential mechanism that leads to the release of DNA/RNA into the circulation, studies have demonstrated that indeed increased amounts of plasma DNA and RNA could be detected in patients sustaining acute traumatic injuries. The degree of plasma DNA elevation correlated with the severity of injury. Similarly, plasma DNA concentrations have been shown to correlate with indices of prognostic significance in patients with acute stroke. It is expected that new diagnostic markers based on plasma RNA detection could be developed for the evaluation of acute pathologies.

Genomic characterisation of the severe acute respiratory syndrome coronavirus of Amoy Gardens outbreak in Hong Kong.

Severe acute respiratory syndrome (SARS) is a global health concern. In Hong Kong, two major outbreaks, one hospital based and the other in the Amoy Gardens apartments, were identified. The frequency of diarrhoea, admission to intensive care, and mortality differed significantly between the two outbreaks. We did genomic sequencing for viral isolates from five Amoy Gardens patients. The virus sequence was identical in four of these five patients. The sequence data from one hospital case and the four identical community cases had only three nucleotide differences. Alterations in the SARS coronavirus genome are unlikely to have caused the distinctive clinical features of the Amoy Gardens patients, and these results highlight the importance of non-viral genomic factors in this outbreak.

Evaluation of a new handheld biosensor for point-of-care testing of whole blood beta-hydroxybutyrate concentration.

OBJECTIVES: To evaluate performance characteristics of the newly available handheld combined glucose and ketone meter for beta-hydroxybutyrate measurement. DESIGN: Laboratory method evaluation. MAIN OUTCOME MEASURES: Accuracy of beta-hydroxybutyrate measurement and effect of acetoacetate interference at clinically important beta-hydroxybutyrate levels. RESULTS: Deming regression analysis of beta-hydroxybutyrate measurements assessed by the ketone sensor and a laboratory enzymatic method revealed a coefficient of determination of 0.989 (P<0.001). Passing-Bablok regression analysis showed a linear relationship between the two methods, ie Y= -0.32+1.13X. The 95% confidence interval of the slope and y-intercept were: slope=1.13 (95% confidence interval, 1.04 to 1.22); intercept= -0.32 (95% confidence interval, -0.59 to -0.06). The Bland-Altman plot showed a small proportional bias between the two methods. The mean bias +/-2 standard deviations was between -0.53 and 0.67 mmol/L. Beta-hydroxybutyrate measurements made by the sensor were linear up to 6 mmol/L. Replicate analysis of two samples spiked with 3.6 mmol/L and 0.8 mmol/L of beta-hydroxybutyrate resulted in coefficients of variation of 3.3% and 13%, respectively. The presence of acetoacetate caused a negative interference in beta-hydroxybutyrate measurement. Beta-hydroxybutyrate recovery was 97.0% and 90.7% when the ketone body ratios were 6:1 and 3:1, respectively. CONCLUSION: The analytical performance of the sensor, when operated according to manufacturer's instructions, could meet the needs of point-of-care beta-hydroxybutyrate measurement. Additional clinical studies are needed to assess the benefits of introducing such an assay in a clinical setting.

Technical optimization of RhD zygosity determination by real-time quantitative polymerase chain reaction: implication for fetal RhD status determination by maternal plasma.

We have recently reported the development of a multiplex, real-time quantitative polymerase chain reaction (PCR) assay for RhD zygosity determination based on the coamplification and quantification of RHD-specific sequences in relation to a reference gene, albumin. This paper discusses the optimization procedure and technical parameters of this multiplex assay.

Simvastatin increases serum osteocalcin concentration in patients treated for hypercholesterolaemia.

Animal studies, experimental models on cell lines, and epidemiological case-control studies have all suggested the possibility that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors have a beneficial effect on bone metabolism. However, all epidemiological studies are not prospective in nature and based on either measurement of bone mineral density or fracture risk. They also differ in recruitment criteria, definition of statin exposure, and outcome assessment. We performed a first prospective study using specific biochemical bone markers on 17 hypercholesterolaemic non-osteoporotic subjects treated with a therapeutic dose of simvastatin 20 mg daily for 4 weeks. Our results show that serum osteocalcin concentration increased significantly (p-value < 0.05) 4 weeks after therapy, whereas other bone markers including serum bone-specific alkaline phosphatase activity, urine deoxypyridinoline, and urine cross-linked N-telopeptides of type I collagen did not show any significant changes. Our data support that simvastatin causes a beneficial effect on bone metabolism as reflected by an increase in serum osteocalcin concentration. This added beneficial effect of statins on bone metabolism could potentially allow statins to become the first effective anabolic agent for the treatment of osteoporosis. We urge that priority should be given to a randomised controlled study to re-evaluate this group of drugs.

Effects of blood-processing protocols on fetal and total DNA quantification in maternal plasma.

BACKGROUND: Recently, apoptotic cells have been found in plasma obtained by centrifugation of blood from pregnant women, raising the question of what constitutes plasma and whether plasma is truly cell free. We compared the effects of different blood-processing protocols on the quantification, DNA composition, and day-to-day fluctuation of fetal and total DNA in maternal plasma. METHODS: Blood samples were collected from healthy pregnant women. The blood sample from each individual was simultaneously processed by different means, including the following: Percoll separation, centrifugation, microcentrifugation, and filtration. The resulting plasma aliquots were subjected to real-time quantitative amplification of the beta-globin (for total DNA) and SRY (for fetal DNA) genes. The differences in the beta-globin and SRY DNA concentrations and the degree of variation between the various plasma aliquots were assessed statistically. RESULTS: Different protocols of blood processing significantly affected the quantification and the day-to-day fluctuation of total (P <0.001), but not fetal (quantification, P = 0.336; fluctuation, P = 0.206), DNA in maternal plasma. The quantitative difference could be attributed to the fact that efficacies of different protocols for generating cell-free plasma vary. Processing blood samples by centrifugation followed by filtration or microcentrifugation is effective in producing cell-free plasma. CONCLUSIONS: Standardization in plasma-processing protocols is needed for maternal plasma DNA analysis, especially for quantification of total DNA in maternal plasma. Such preanalytic factors may also affect other applications of plasma DNA analysis.

Determination of RhD zygosity: comparison of a double amplification refractory mutation system approach and a multiplex real-time quantitative PCR approach.

BACKGROUND: Rh isoimmunization and hemolytic disease of the newborn still occur despite the availability of Rh immunoglobulin. For the prenatal investigation of sensitized RhD-negative pregnant women, determination of the zygosity of the RhD-positive father has important implications. The currently available molecular methods for RhD zygosity assessment, in general, are technically demanding and labor-intensive. Therefore, at present, rhesus genotype assessment is most commonly inferred from results of serological tests. The recent elucidation of the genetic structure of the prevalent RHD deletion in Caucasians, as well as the development of real-time PCR, allowed us to explore two new approaches for the molecular determination of RhD zygosity. METHODS: Two methods for RhD zygosity determination were developed. The first was based on the double Amplification Refractory Mutation System (double ARMS). The second was based on multiplex real-time quantitative PCR. For the double ARMS assay, allele-specific primers were designed to directly amplify the most prevalent RHD deletion found in RhD-negative individuals in the Caucasian population. The multiplex real-time quantitative PCR assay, on the other hand, involved coamplification and quantification of RHD-specific sequences in relation to a reference gene, albumin, in a single PCR reaction. A ratio, DeltaCt, based on the threshold cycle, was then determined and reflects the RHD gene dosage. RESULTS: The allele-specific primers of the double ARMS assay reliably amplified the RHD-deleted allele and therefore accurately distinguished homozygous from heterozygous RhD-positive samples. The results were in complete concordance with serological testing. For the multiplex real-time quantitative PCR assay, the DeltaCt values clearly segregated into two distinct populations according to the RHD gene dosage, with mean values of 1.70 (SD, 0.17) and 2.62 (SD, 0.29) for the homozygous and heterozygous samples, respectively (P: <0.001, t-test). The results were in complete concordance with the results of serological testing as well as with the double ARMS assay. CONCLUSION: Double ARMS and real-time quantitative PCR are alternative robust assays for the determination of RhD zygosity.

Off-line coupling of capillary electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

An automated fraction collection interface is used in conjunction with matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry to analyze material isolated by capillary electrophoresis (CE). CE fractions are deposited directly on the MALDI probes so that individual peaks from the electropherogram are associated with a single sample spot on the probe. MALDI matrices with high acid concentrations afford enhanced tolerance of electrophoresis buffers. The utility of this hybrid instrument is demonstrated by separation and mass analysis of a tryptic digest of cytochrome c and synthetic mixtures of four proteins. Mass assignments corresponding to the protonated molecular ions are in good agreement with those predicted from molecular structure. Miniaturization of the interface affords enhanced sensitivity, with good-quality spectra from separations of as little as 25 fmol of protein.

Nuclear DNA polymerases and the HeLa cell cycle.

Purified nuclei of HeLa S3 cells contain two DNA-dependent DNA polymerases that have distinct physical and enzymatic properties. We have investigated the variations in their activity during the cell cycle of a synchronized culture. Cells were synchronized by a double thymidine block, harvested at various phases of the cycle, and the two DNA polymerases were purified partially by DEAE-cellulose and phosphocellulose chromatography. The activity of DNA polymerase I (low molecular weight, N-ethylmaleimide-insensitive) remains essentially constant throughout the cycle. The activity of DNA polymerase II (high molecular weight, N-ethylmaleimide-sensitive), however, increases during G1 to mid-S and declines, 7- to 10-fold between late-S and G2. Addition of cycloheximide (60 mug/ml) to cultures 12 hours after the release from thymidine block abolishes the rise in the activity of DNA polymerase II. Cycloheximide also reduced the activity of DNA polymerase I by 60%. Addition of hydroxyurea (1mM) at 1 hour after release has no effect on the activity of either enzyme. We conclude that in HeLa cells, DNA polymerase I and II are distinct enzymes, that DNA polymerase II probably functions in DNA replication and is probably induced in response to stimuli for DNA biosynthesis.