BMC Cancer 2008, 8:213 http://www.biomedcentral.com/1471-2407/8/213 by cycling temperature capillary electrophoresis (CTCE), with mean separating temperatures of 48.5°C and ampli- tudes of 3°C cycled 20 times. K-ras mutations in exon 1 were identified by co-analysis with a mutated internal standard in a similar manner as previously described by Bjørheim et al. [20]. Additionally, samples with a high mutant fraction were sequenced according to standard DNA sequencing protocol (GE Healthcare Bio-Sciences AB). Follow-up The patients were followed for five years with X-ray of the chest and CT of the pelvis and abdomen every three to six months for five years or to the end of the study period. Data concerning death was crosschecked with the national health register. Statistics Patient's data were prospectively registered in a database. All data was analyzed using the Statistical Package SPSS 12.5 for Windows. The chi-squared tests were used for sta- tistical analysis and differences were considered signifi- cant at P < 0.05. For survival analysis, Kaplain-Meiers curves were constructed, and using the log-rank test assessed differences in survival between groups. Results The clinical pathological characteristics of the 237 rectal cancer patients are summarized in Table 1. Of the eleven possible risk factors tested with regard to patients with positive and negative k-ras markers in the lavage fluid, only N- and R-stage were significantly different between the two groups (p = 0.03 and p = 0.002, respectively). This was mainly due to a higher percentage of N0 and R0 stages in the k-ras negative group. We analyzed 186 of the removed tumours for k-ras mutations and 56 of these were k-ras positive. 19 out of 237 patients had a positive k-ras marker in the lavage fluid (Table 2). Of the 19 cases with k-ras positive lavage there were 12 R0, 3 R1 and 4 R2. We have estimated survival rate for the patients with positive and negative marker for lavage fluid by a Kaplan-Meier plot (Figure 1) and a log rank test. Mean observation time was 25 (1–66) months for all patients. Patients positive for the marker in the lavage fluid had a mean survival of 22 months with a Std. Error 3.7 (C.I 14.6–29.1) months compared to 47 months with a Std. Error 2.4 (C.I 43.2– 50.8) for patients with a negative marker (p = 0.006). As R1 and R2 resections are more likely to have positive lav- age fluid a new log rank test was done with only R0 patients. The result showed that there was still a signifi- cant difference between the two groups (p = 0.02). There was no significant difference (p = 0.3) between k-ras positive and negative tumours with respect to survival (Figure 1b). Likewise when mutational status of k-ras for A B tFAoi.gKsuu-rravesiv1ianl lavage samples immediately after surgery related A. K-ras in lavage samples immediately after surgery related to survival. B. K-ras in tumours samples from rectal cancers patients related to survival. the tumour was compared for R0, the non-significant role was maintained (p = 0.09). In two of the cases of R0 resections with k-ras positive lav- age the preoperative biopsy was classified as k-ras negative being either a result of non-representative biopsy or a multi-clonal locally advanced tumour. Page 3 of 8 (page number not for citation purposes)