BRAF V600E is the predominantly occurring mutation of the cytoplasmic kinase BRAF , and , in colorectal cancer , its determination provides a diagnostic exclusion criterion for hereditary nonpolyposis colorectal cancer. The aim of our study was to develop a sensitive BRAF V600E high resolution melting (HRM) assay. We first established and optimized the BRAF HRM assay using a cell line dilution model , enabling us to detect 1% mutant DNA in a background of wild-type DNA. In a comparison , DNA sequencing and real-time allele-specific PCR in the cell line dilution model HRM assay proved to be more sensitive than DNA sequencing and denaturing high performance liquid chromatography , retaining the same sensitivity as real-time allele-specific PCR. In a learning set of 13 patients with known BRAF V600 status , the mutation was detected with high concordance by all four methods. Finally , we validated the HRM assay on 60 formalinfixed , paraffin-embedded colorectal cancer samples. Although all mutated samples were correctly identified by HRM , the detection limit of the HRM assay decreased when using low-quality DNA derived from formalin-fixed , paraffin-embedded samples. In conclusion , HRM analysis is a powerful diagnostic tool for detection of BRAF V600E mutation with a high sensitivity and high-throughput capability. Despite the expected decrease in sensitivity , HRM can reliably be applied in archival formalin-fixed , paraffin-embedded samples tissues.

Patients suffering from severe constipation are a new challenge for sacral nerve stimulation but further research on pelvic floor function is needed.

SNS is of value in selected patients with neurogenic faecal incontinence.

Toxic colitis complicated by toxic megacolon can occur after various diseases of the colon and remains a life-threatening disorder associated with a significant risk of postoperative complications. Subtotal colectomy with ileostomy remains the procedure of choice. Surgical colonic decompression with faecal diversion alone is associated with a high rate of complications.

Recently, evidence has emerged indicating that assessment of KRAS mutations before anti-epidermal growth factor receptor therapy improves outcome in patients with metastatic colorectal cancer (CRC). We report here a novel reverse-hybridization (RH) assay to screen for KRAS mutations in formalin-fixed paraffin-embedded colorectal tissue samples. We combined mutant-enriched PCR based on peptide nucleic acid clamping and RH of amplification products to nitrocellulose test strips that contained a parallel array of oligonucleotide probes targeting 10 frequent mutations in codons 12 and 13 of the KRAS gene. DNA mixing experiments, which included eight different tumor cell lines with known KRAS mutations, were performed to examine the sensitivity of mutation detection. All KRAS mutations present in tumor cell lines were unambiguously identified by the RH assay with 1% of each cell line DNA diluted in normal DNA. RH was then used to screen for KRAS mutations in 74 colorectal tumor and 4 normal control samples. Twenty-six (35%) of the 74 tumor samples showed KRAS mutations. No mutation was found in the four samples of normal colorectal tissue. DNA sequencing without previous mutant enrichment, however, failed to detect four (15%) out of 26 KRAS-positive formalin-fixed paraffin-embedded samples (FFPE). This finding suggests that even after microdissection, mutant sequences in a given DNA isolate can be rare and more sensitive methods are needed for mutation analysis.