BMC Cancer 2008, 8:213 http://www.biomedcentral.com/1471-2407/8/213 pelvic lavage after resection of rectal cancer was hypothe- sized to be of prognostic value. One of the most promi- nent mutated genes in colorectal cancer is Kirsten ras 2 (k- ras), a member of the RAS family. Mutations in the k-ras gene is described as an early event in the process of color- ectal carcinogenesis [11]. K-ras mutations are mainly found in exon 1, codon 12, 13 and exon 2, codon 61. Mutations in codon 12 account for 80–90% of k-ras gene alterations with several hotspots [12]. Oncogenic muta- tions of k-ras are involved in 20–50% of colorectal cancers [13,14]. The k-ras mutation frequency in rectal cancer has been reported to be 15–33% which is lower than in colon cancer [15,16]. We used k-ras exon 1 as the only tumour cell-associated marker to determine the presence of dis- seminated tumour cells in peritoneal lavage samples from patients undergoing surgery for rectal cancer. The aim of this pilot study was to evaluate the method denaturant capillary electrophoresis (DCE), to detect tumour cells in peritoneal lavage fluid of patients undergoing resection for rectal cancer. Methods Study population The University Hospital Rikshospitalet-Radiumhospitalet is a tertiary referral center for locally advanced primary and recurrent rectal cancer. The treatment regime is multi- modal and includes preoperative radiotherapy or chemo- radiation. Our patients undergo surgery a median 56 days after end of long course radiation therapy. Peritoneal lavage from 237 patients and tumour samples from 186 patients (59% men, 41% women) with either locally advanced or local recurrent rectal cancer was col- lected during surgery at the Norwegian Radium Hospital between 2000 and 2006. Patient's age ranged from 29 to 87 years with a median age of 66. All samples were col- lected after written informed consent. Possible risk factors for dissemination of cancer cells intraoperatively were reg- istered: resection through the periphery of the cancer as in R1 and R2 resections, tumour perforation or the presence of lymph node metastases in the mesorectum. Penetration of cancer through the serosa and the presence of macro- scopic peritoneal carsinomatosis may of course give rise to free cancer cells and were thoroughly registrated. Two cases with registrated peritoneal carsinomatosis were tested as "positive controls". Tumours were pathologically staged according to the TNM classification [17] except for the local recurrences that do not fit into this except for pTo. This is due to the observation that a recurrence usu- ally starts outside the rectal wall and most often there are no lymph nodes to resect [18]. The local surgical achieve- ments were staged as follows: R0 microscopically free cir- cumferential and distal margins, R1 microscopically involved margins, and R2 locally macroscopic residual cancer or no resection. N0; No regional lymph node metastasis. N1; Metastasis in 1 to 3 regional lymph nodes. N2; Metastasis in 4 or more regional lymph nodes. NX; unknown status of lymph nodes. Sample treatment After resection of the tumour the pelvis was washed with sterile water 200–600 ml (discarded) subsequently fol- lowed by 200–600 ml saline water. Thereafter two speci- mens of 50 ml were aspirated to 50 ml centrifuge tubes. The second sample of 50 ml most often contained more white blood cells than the first sample because haemosta- sis was not complete. Cells were harvested by centrifuga- tion at 1200 g for 10 minutes followed by removal of the supernatant. The cell pellets were frozen at -20°C until DNA extractions were performed. QIAamp DNA Kit (Qia- gen, Valencia, California, USA) was used for the DNA extraction, following the manufacturers instructions. Polymerase chain reaction The target sequences were amplified through a nested PCR protocol to circumvent amplification of a pseudogene. Primers were designed with use of Primer 3 [18,19]. The first PCR reaction consisted of approximately 50 ng genomic DNA, 0.05 U/μl Taq, polymerase, 0.005 U/μl PFU polymerase, 1× Buffer (ABgene), 2.5 mM MgCl2, and 0.4 mM dNTP mix (ABgene) and 0.15 μM of each primer (5'CTTAAGCGTCGATGGAGGAG3', 5'AGAATGGTCCTGCACCAGTAA3'). The second PCR used a 1:1000 dilution of the first PCR products as tem- plate while the polymerase, buffer, MgCl2 and dNTP con- centrations stayed the same. Primers, one labelled primer with GC-clamp (5'- 6Fam-CGCCCGCCGCGCCCCGCGC CCGTCCCGCCGCCCCCGCCCGCCTCTATTGTTGGATC ATATTC3') at a final concentration of 0.11 μM and one reveres primer (5'CATTATTTTTATTATAAGG3') in a final concentration 0.3 μM were added to the mix. The total volumes of both PCR reactions were adjusted to 10 μl with sterile water. Temperature cycling was performed in a Mastercycler® (Eppendorf, Bergman AS, Lillestrøm, Nor- way) using the following cycling conditions: denaturation 5 min at 96°C, followed by 35 cycles of 95°C in 30 s, annealing 56°C in 30s (first PCR) and 48°C in 30s (sec- ond PCR) and 72°C in 60s. We also analysed k-ras exon 2, and braf exon 11 and exon 15, but were unable to find any mutations with these markers in the lavage samples. Mutation detection Amplified 6-fam labelled PCR products were analysed by denaturant capillary electrophoresis in a MegaBACE 1000 DNA Analysis System (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The base substitutions were separated Page 2 of 8 (page number not for citation purposes)