Molecular detection (k-ras) of exfoliated tumour cells in the pelvis is a prognostic factor after resection of rectal cancer?

<p>Abstract</p> <p>Background</p> <p>After total mesorectal excision (TME) for rectal cancer around 10% of patients develops local recurrences within the pelvis. One reason for recurrence might be spillage of cancer cells during surgery. This pilot study was conducted to investigate the incidence of remnant cancer cells in pelvic lavage after resection of rectal cancer. DNA from cells obtained by lavage, were analysed by denaturing capillary electrophoresis with respect to mutations in hotspots of the <it>k-ras </it>gene, which are frequently mutated in colorectal cancer.</p> <p>Results</p> <p>Of the 237 rectal cancer patients analyzed, 19 had positive lavage fluid. There was a significant survival difference (p = 0.006) between patients with <it>k-ras </it>positive and negative lavage fluid.</p> <p>Conclusion</p> <p>Patients with <it>k-ras </it>mutated cells in the lavage immediately after surgery have a reduced life expectation. Detection of exfoliated cells in the abdominal cavity may be a useful diagnostic tool to improve the staging and eventually characterize patients who may benefit from aggressive multimodal treatment of rectal cancer.</p

Detection of Mutations in Exon 8 of TP53 by Temperature Gradient 96-Capillary Array Electrophoresis

Various capillary electrophoresis applications have increasingly been utilized in mutation detection. Separation of two species is either based on secondary structure or differences in melting of DNA due to the mutation. Detection of the mutant is based on its mobility difference in the sieving matrix. We have adapted a regular 96-capillary sequencing instrument, the MegaBACETM 1000, for mutation detection based on thermodynamic stability and mobility shift during electrophoresis. Denaturation of the lower melting domain of the DNA was achieved with a gradually decreasing temperature gradient in combination with a chemical denaturant. Samples were analyzed for mutants in exon 8 of the TP53 gene from tumor samples and controls. Genomic DNA was PCR-amplified with one fluorescein labeled primer and one GC-clamped primer, diluted in water, and analyzed by temperature gradient 96-capillary array electrophoresis. Tumor samples and PCR reconstruction experiment samples were resolved by capillary gel electrophoresis under appropriate temperature gradient denaturing conditions. Ninety-six samples were analyzed in one run, with an analysis time of 30 min and a sensitivity to detect mutated alleles in wild-type background down to 0.4%. The technique proved to be robust, in that the gradient compensates for temperature differences within the capillary chamber; thus, each capillary will pass through the optimal separating conditions around the theoretical melting temperature for TP53 exon 8, separating homoduplexes and heteroduplexes. This technique is applicable to any sequence previously analyzed by DNA melting gel techniques or sequences harboring iso-melting domains of 100–V120 bp

Automated Constant Denaturant Capillary Electrophoresis Applied for Detection of KRAS Exon 1 Mutations

In this study, we have applied automated constant denaturant capillary electrophoresis (ACDCE) for the detection of KRAS exon 1 mutations. Samples from 191 sporadic colon carcinomas previously analyzed for KRAS mutations with allele-specific PCR (ASPCR), temporal temperature gradient electrophoresis (TTGE), and constant denaturant capillary electrophoresis (CDCE) were analyzed. In ACDCE, an unmodified ABI PRISM™ 310 genetic analyzer with constant denaturant conditions separated fluorescein-labeled PCR products. Temperature in combination with a chemical denaturant was used for separation. The optimal separation conditions for PCR-amplified KRAS exon 1 fragments were determined by adjusting the temperature before electrophoresis. In the ACDCE analysis, the sequence of a mutant was determined by comparing the electropherogram of the fragment to that of known mutations followed by mixing the sample with control mutations before reanalysis. In a titration experiment mixing mutant and wild-type alleles, the sensitivity for mutation detection was shown to be 0.6% in this automated CDCE technique. The automation of CDCE allowed rapid analysis of a large number of test samples over as short period of time and with a commercially available apparatus