BMC Cancer 2008, 8:213 http://www.biomedcentral.com/1471-2407/8/213 in colorectal cancer patients [22,23]. The higher percent- age of N0 stage in the k-ras negative group could not explain the difference either. In our study there was no sig- nificant difference in estimated survival, but a possible trend observed between patients with k-ras positive and k- ras negative tumor markers in the solid tumor samples. This may rest upon the fact that the plots are more or less similar until 30 months postoperatively and diverge after this observation time. Thus within the observed period a k-ras mutation in the tumor itself does not explain the prognostic differences of the patients. In this study we have found that patients with tumour cells in abdomen after operation tend to have a worse prognosis than patients without free tumour cells. Our results in this study are similar to those of some studies with regard to survival [24-27], but in conflict with other results [28,29]. These differences could be related to patient selection and the methods used for detection as well as the follow-up methods. It has become apparent in recent years that not only do the pathologic characteristics of rectal cancer influence the long-term outcome in terms of local recurrence and sur- vival, but also that the surgeon is an important variable. This previously has been demonstrated by the reports from the Norwegian Rectal Cancer Group [30,31]. Better treatment, specialist care, recruitment into clinical trials or treatment protocols and any combination of these factors almost certainly influence regional variations in cancer outcome. The Norwegian Radium Hospital is a compre- hensive cancer centre with high-volume surgery and the rectal cancers patient population is mostly TNM stage II and III. In this pilot study only rectal cancer patients have been included. The detection of disseminated tumour cells depends on a number of steps including collection and treatment of samples, cell separation protocols and chosen markers. Technical advancements in detecting free DNA have been made over the years. All methods used depend on the rec- ognition of antigens or gene markers assumed to be exclu- sively expressed by tumour cells and not by normal tissue in the examined sample. A cytology based method can only detect nanogram quantities of DNA. Immunohisto- chemistry leads to an increased sensitivity, but without improved specificity. High number of false positives due to non specific labelling was still observed and low sensi- tivity and specificity contribute to the conflicting results previously reported in detection of disseminated tumour cells [25,28,29]. With the introduction of PCR, smaller quantities of DNA could be detected. The robustness of such analysis has been improved by fluorescence-based allelotyping techniques involving capillary electrophore- sis. These molecular detection methods can be divided in two groups: Detection of RNA with marker-gene expres- sion (m-RNA) and detection of RNA specific to the tissue. This is a sensitive method with a possible detection of one single mutated cell in up to 108 wild-type cells in an opti- mal condition. However RNA is unstable in the extracel- lular environment and its detection in tissue or fluids is thus dependent on the presence of viable tumour cells. Routinely performed clinical sampling methods such as lavage may not be capable to preserve the RNA capacity of the cells. The other method is detection of tumour specific chromosomal abnormalities or mutations. In solid tumours these variations are heterogenous and complex with a need for detecting mutations in a small fraction. We have previously demonstrated that the DCE method used in this study fulfils these criteria with a sensitivity of 4 × 10-3 [32]. The viability of exfoliated cells and the ability of these cells to become implanted and proliferated in the perito- neum have been confirmed by several studies [33,34]. We have not tested the viability of the tumour cells, because we assume that they are all compromised after the lavage treatment of sterilized water. A high diagnostic certainty is essential for establishing microscopic peritoneal dissemi- nation as a prognostic factor. A standard procedure of peritoneal washing with sterile water subsequently fol- lowed by saline water was done after operation. Given the procedure and the sensitivity of the k-ras assay (limit is reported to be 1% for the homoduplexes and 0.1% for the heteroduplexes for a varity of target sequences), minor or moderate bleeding in the surgical area could result in a mutant fraction below the detection limit. One microliter of blood contains an average of 5000 white blood cells, hence if 10 millilitre of blood leaked into the lavage area, 50 000 k-ras positive tumour cells would be required for detection. Albeit this stringent limitation, k-ras mutant positive cells were regularly detected in lavage fluids from the pelvic cavity. There are no known tumour-specific markers for rectal cancer. K-ras is shown to be up-regu- lated in epithelial tumour cells [35] and was used as a marker for free tumour cells or cell remnants in the peri- toneal cavity. The k-ras gene has a small hotspot region, which makes it possible to detect 80–90% of k-ras gene alterations with a simple PCR. However variation (20– 50%) of the prevalence of k-ras mutations in small spo- radic colorectal adenomas have been reported in the liter- ature [35]. Specific k-ras mutations induce different biological consequences by affecting differently the struc- tural confirmation and the function of the mutated pro- tein [36,37]. For example, a number of studies have demonstrated an impact of k-ras mutations for cancer pro- gression and predispose to more aggressive biological behaviour in patients with colorectal cancer [36,38-40]. K-ras mutations have also shown to be predictor of resist- ance to the anti-epidermal growth factor receptor (cetuxi- Page 6 of 8 (page number not for citation purposes)