Genome-wide, high-resolution detection of copy number, loss of heterozygosity, and genotypes from formalin-fixed, paraffin-embedded tumor tissue using microarrays

Formalin-fixed, paraffin-embedded (FFPE) material tends to yield degraded DNA and is thus suboptimal for use in many downstream applications. We describe an integrated analysis of genotype, loss of heterozygosity (LOH), and copy number for DNA derived from FFPE tissues using oligonucleotide microarrays containing over 500K single nucleotide polymorphisms. A prequalifying PCR test predicted the performance of FFPE DNA on the microarrays better than age of FFPE sample. Although genotyping efficiency and reliability were reduced for FFPE DNA when compared with fresh samples, closer examination revealed methods to improve performance at the expense of variable reduction in resolution. Important steps were also identified that enable equivalent copy number and LOH profiles from paired FFPE and fresh frozen tumor samples. In conclusion, we have shown that the Mapping 500K arrays can be used with FFPE-derived samples to produce genotype, copy number, and LOH predictions, and we provide guidelines and suggestions for application of these samples to this integrated system.     

Genetic pathways and mutation profiles of human cancers: site- and exposure-specific patterns

Cancer is a complex disease that involves the accumulation of both genetic and epigenetic alterations of numerous genes. Data in the Genetic Alterations in Cancer database for gene mutations and allelic loss [loss of heterozygosity (LOH)] in human tumors (e.g. lung, oral, esophagus, stomach and colon/rectum) were reviewed. Results for the genes and pathways implicated in tumor development at these sites are presented. Mutation incidence, spectra and codon specificity are described for lung, larynx and oral tumors. LOH occurred more frequently than gene mutations in tumors from all sites examined. The cell cycle gene, TP53 (all sites), and cell signaling gene, APC (colorectal and gastric cancers), were the only genes with similar incidences of LOH and mutation. Alterations of one or more cell cycle and cell signaling genes were reported for tumors from each site. Site-specific activation was apparent in the cell signaling mitogen-activated protein kinase oncogenes (KRAS in lung, HRAS in oral cancers and BRAF in esophageal and colorectal cancers). Analysis of genetic changes in lung tumors showed that the incidence of mutations in the TP53 and KRAS genes and the incidence of LOH in the FHIT gene were significantly greater in smokers versus non-smokers (P < 0.01). In lung and oral cancers, the TP53 GC --> TA transversion frequency increased with tobacco smoke exposure (P < 0.05). Furthermore, the TP53 mutational hot spots for lung and laryngeal cancers from smokers included codons 157, 245 and 273, whereas for oral tumors included codons 280 and 281.     

Spinal anaesthesia for Caesarean section in a patient with systemic sclerosis

We describe the management of a diabetic primigravid woman with systemic sclerosis and thrombocytopaenia who required Caesarean section for pre-eclampsia. This was performed successfully under spinal anaesthesia.     

Procedural Complications During Congenital Cardiac Catheterization

Objective. We sought to identify complications that occurred during congenital cardiac catheterization (CCC) and determine factors that could improve the quality of care provided to patients with congenital heart disease during this procedure. Design. We reviewed the electronic medical record for 903 CCC cases, (455 female; mean age = 29 ± 22 years, range = birth to 91 years) performed in our catheterization laboratory from 2005 to 2007. Included in this cohort are 342 cases performed on patients less than 18 years of age. Clinical follow-up data were reviewed for 3 months postcatheterization. Complications were assigned a grade from 1 to 4 based on severity. Results. The indication for catheterization was diagnostic in 459 (51%) patients, interventional in 386 (43%) patients, and endomyocardial biopsy in 58 (6%) patients. Mean intravenous contrast dose = 1.9 ± 1.8 mL/kg. Mean fluoroscopy exposure = 22 ± 13 minutes. Mean procedure duration = 122 ± 42 minutes. Although 806 cases (89%) were performed without complication, 102 complications were observed in 97 cases. There were no deaths. Emergent surgery was performed in four patients. One patient notified us 16 days after catheterization that she was pregnant. The result of that pregnancy was normal. Thirty complications occurred during the CCC procedure and a first year fellow was involved in 17. Patient age, weight, gender, attending physician, or type of procedure (diagnostic vs. intervention) did not impact risk of complications. Conclusions. Patients of all ages with congenital heart disease can expect a safe procedure with minimal risk of serious complications. Procedural changes that have been implemented include pregnancy testing on all menstruating females prior to CCC regardless of history of sexual activity, and first-year fellows are now directly supervised by the attending physician rather than a more senior fellow throughout the procedure.     

Program to remove incorrect allergy documentation in pediatrics medical records.

The incidence of incorrectly reported drug allergies in a pediatrics hospital and the effectiveness of pharmacist interventions to clarify these reports were studied. A four-month prospective study included children (< or = 18 years of age) with at least one drug allergy reported in their medical chart. Drug allergies were assessed by a pharmacist who labeled the reactions as true, incorrectly reported, or undetermined allergies, in accordance with defined criteria. When an incorrectly reported allergy was removed from a patient's chart with the consent of the attending physician, the intervention was reported to the community pharmacist. A total of 186 of 248 drug allergies identified in 1591 patient charts were challenged. Of these, 26 (14%), 103 (55%), and 57 (31%) were considered true, undetermined, and incorrectly reported drug allergies, respectively, by the pharmacist. A total of 53 (93%) incorrectly reported allergies were removed from patients' charts with the consent of the attending physicians. Community pharmacists were contacted in 25 of these cases. At follow-up, the incorrect allergy documentation was found to have been removed from 23 community pharmacy charts. A pharmacist found numerous incorrectly reported allergies in a pediatrics hospital and assisted in removing them from patients' medical charts.     

Salmonella in Broiler Litter and Properties of Soil at Farm Location

Contamination of litter in a broiler grow-out house with Salmonella prior to placement of a new flock has been shown to be a precursor of the flock's Salmonella contamination further down the production continuum. In the southern USA, broiler grow-out houses are primarily built on dirt pad foundations that are placed directly on top of the native soil surface. Broiler litter is placed directly on the dirt pad. Multiple grow-out flocks are reared on a single litter batch, and the litter is kept in the houses during downtime between flocks. The effects of environmental determinants on conditions in broiler litter, hence Salmonella ecology within it, has received limited attention. In a field study that included broiler farms in the states of Alabama, Mississippi and Texas we assessed Salmonella in broiler litter at the end of downtime between flocks, i.e. at the time of placement of a new flock for rearing. Here we utilized these results and the U.S. General Soil Map (STATSGO) data to test if properties of soil at farm location impacted the probability of Salmonella detection in the litter. The significance of soil properties as risk factors was tested in multilevel regression models after accounting for possible confounding differences among the farms, the participating broiler complexes and companies, and the farms' geographical positioning. Significant associations were observed between infiltration and drainage capabilities of soil at farm location and probability of Salmonella detection in the litter.     

From the Cover: The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake

Numerous clinical trials using folic acid for prevention of cardiovascular disease, stroke, cognitive decline, and neural tube defects have been completed or are underway. Yet, all functions of folate are performed by tetrahydrofolate and its one-carbon derivatives. Folic acid is a synthetic oxidized form not significantly found in fresh natural foods; to be used it must be converted to tetrahydrofolate by dihydrofolate reductase (DHFR). Increasing evidence suggests that this process may be slow in humans. Here we show, using a sensitive assay we developed, that the reduction of folic acid by DHFR per gram of human liver (n = 6) obtained from organ donors or directly from surgery is, on average, less than 2% of that in rat liver at physiological pH. Moreover, in contrast to rats, there was almost a 5-fold variation of DHFR activity among the human samples. This limited ability to activate the synthetic vitamer raises issues about clinical trials using high levels of folic acid. The extremely low rate of conversion of folic acid suggests that the benefit of its use in high doses will be limited by saturation of DHFR, especially in individuals possessing lower than average activity. These results are also consistent with the reports of unmetabolized folic acid in plasma and urine.