Patient characteristics are summarized in Table 1. The median age of the patients included in this study was 65 y (181 women, 92 men). Most underwent primary tumor resection (260/273 patients, 95.2%), while secondary metastatic disease was diagnosed in 194 patients (71.1%), of whom 70 (36.1%) underwent resection and 22 (11.3%) underwent thermoablation. The primary tumor was located in the colon in 112 patients (41.1%), sigmoid colon in 100 patients (36.6%) and rectum in 61 patients (22.3%). The metastases were located in the liver in 129 (66.5% of patients with metastases), in the lungs in 39 (20.1%), and in other organs in 126 patients (64.9%). Pretreatment carcinoembryonic antigen (CEA) levels were elevated above the normal range in 89 patients (32.6%).

K-Ras gene mutations were present in 89 patients (32.6%), of whom 76 (85.4%) had mutations in codon 12 and 10 (11.2%) had mutations in codon 13. Women showed a higher incidence of K-Ras gene mutations relative to men (p = 0.0290). No significant differences were observed with respect to tumor size, lymph node involvement grade, histological grade, histopathological type, primary tumor localization, performance status, age, or pretreatment CEA level.

B-Raf gene mutations were present in 17 patients (6.9%), of whom 6 (35.3%) had mutations in exon 15. One patient had a mutation in exon 11, while mutation status was not determined in 10 patients (58.8%). A higher incidence of B-Raf gene mutations was detected in patients with low-grade neoplasm (p < 0.0001), primary tumor localization outside the sigmoid colon (p = 0.0467) and with non-tubular neoplasms (p = 0.0468). Other parameters assessed were not statistically different.

There were no significant differences in OS rates between patients with K-Ras mutations and wild-type K-Ras genes (p = 0.6869; Fig. 1). A perceptible trend to prolongation of OS was apparent when K-Ras mutations were present in codon 13 relative to codon 12 (p = 0.0830; Fig. 2).

Similarly, mutations in the B-Raf gene showed no prognostic significance (Fig. 3). Patients with disseminated CRC (M+) and B-Raf gene mutations tended toward shorter OS relative to those with wild-type B-Raf genes (p = 0.06723).

These results are summarized in Table 2. Univariate analysis identified the following prognostic factors as influencing OS rate in this patient cohort: Age, patients 75 y and older lived for 36.7 mo relative to those younger than 75 (58.9 mo) (p = 0.0472); Gender, female patients lived for 62.7 mo relative to 42.6 mo for male patients (p = 0.0328); Primary tumor localization, patients with primary tumors in the sigmoid colon lived for 68.0 mo compared with 43.5 mo in patients with primary tumors located in the colon or rectum (p = 0.0039); Performance status, patients with a good performance score e.g., WHO 0–1 (58.4 mo) and Karnofsky status 81–100% (58.1 mo) lived longer relative to those with poor performance status (19.0 and 19.4 mo for patients with WHO 2–3 and Karnofsky status ≤ 80%, respectively) (p = 0.0027 and p = 0.0036, respectively); Lymph node involvement grade, survival in patients without lymph node metastases was 65.3 mo relative to 46.3 mo in patients presenting with metastases (p = 0.0031); Pretreatment CEA level, median time to progression in patients with normal pretreatment CEA level ≤ 5 ng/ml was 76.3 mo relative to 25.6 mo in patients with increased pretreatment CEA level (p < 0.0001; Table 2).

Other clinical parameters such as histological differentiation grade and primary tumor size showed no significant differences between groups.

These results are summarized in Table 2. Multivariate analysis identified the following independent prognostic factors affecting OS rates: Primary tumor localization (HR 0.53; p = 0.0032); Pretreatment CEA level (HR 2.68; p < 0.0001); WHO performance status (HR 0.34; p = 0.0008); Lymph node involvement grade (HR 1.94; p = 0.0107).

Other clinical parameters such as age, gender, and Karnofsky performance status showed no significant differences in this analysis.

These results are summarized in Table 3. Patients with higher pretreatment levels of CEA (> 5 ng/ml) showed a median time to progression of 9.0 mo relative to 13.0 mo in patients with normal levels (≤ 5 ng/ml, p = 0.0085). Patients without resection of metastases showed a median time to progression of 9.0 mo relative to 14.0 mo in patients who underwent resection (p = 0.0131). Patients with K-Ras gene mutations showed a median time to progression of 9.0 mo relative to 11.0 mo in those with the wild-type K-Ras gene (p = 0.05883). Other clinical parameters including histological differentiation grade, primary tumor location and size, lymph node involvement grade, and B-Raf gene mutation status showed no predictive significance in this analysis.

These results are summarized in Table 3. Multivariate analysis identified the following independent favorable predictive factors in patients with disseminated CRC treated with irinotecan-based first-line palliative chemotherapy: Wild-type K-Ras gene (HR 0.59; p = 0.0459) and normal pretreatment CEA levels (HR 0.52; p = 0.0065).

However, this analysis did not reveal any significant differences between patients with and without resection of metastases, with different histological types of neoplasms and B-Raf gene mutation status.

These results are summarized in Table 4. Univariate analysis of time to progression in patients treated with oxaliplatin-based first-line chemotherapy regimens reveals that increased CEA levels and resection of metastases exerted significant influences on median time to progression. Patients with increased pretreatment CEA levels had a time to progression of 8.0 mo compared with 13.0 mo in patients with normal CEA levels (p = 0.0084). Patients without resection of metastases had a time to progression of 9.0 mo relative to 16.0 mo in patients who underwent resection (p = 0.0226). Patients with tubular tumors showed a time to progression of 9.0 mo compared with 13.0 mo in those with other histological types (p = 0.0462). Patients with K-Ras gene mutations did not show a significant difference in time to progression when treated with oxaliplatin chemotherapy, when compared with those with the wild-type K-Ras gene (Fig. 4). The significance of B-Raf gene status, WHO performance status, and Karnofsky performance status could not be assessed.

These results are summarized in Table 4. Multivariate analysis identified resection of metastases (HR 0.43; p = 0.0249) and wild-type K-Ras gene (HR 0.49; p = 0.0451) as independent favorable predictive factors in patients with disseminated CRC who were treated with oxaliplatin-based first-line palliative chemotherapy regimens.

However, no statistically significant effects of CEA levels and types of neoplasm could be seen. The significance of B-Raf gene status, WHO performance status, and Karnofsky performance status could not be assessed due to the small number of patients.

The research approved by the appropriate local ethical committees (reference numbers: WIM-50/2008 and WIM-45/2009).

273 consecutive patients (median age 65 y, range 25–85 y) with CRC who were treated between 2006 and 2010 at the Oncology Department of the Military Institute of the Heath Services, Warsaw, were included in this study (See Table 1 for an overview of patient characteristics).

Inclusion criteria consisted of a confirmed histopathological diagnosis of colorectal cancer, availability of adequate primary tumor material, and a lack of effect of chemotherapy or radiotherapy on the tumor.

Surgically removed primary tumor tissue specimens were fixed in formalin and converted into paraffin blocks for further analysis.

Primary tumor tissue collected from colorectal cancer patients was fixed in 10% neutral buffered formalin for 24 h and converted into paraffin blocks. Serial 5 µm-thick sections of each paraffin block corresponding to representative areas of the tumors were stained with hematoxylin/eosin (H&E) and the presence of tumor tissue verified by an experienced pathologist.

DNA from paraffin-embedded tissue was prepared from 10–30 µm sections after macrodissection, to ensure they contained at least 80% tumor cells. Tissue samples were extracted with xylene and ethanol to remove paraffin and placed in 1% SDS/proteinase K (10 mg/ml) at 56°C overnight. DNA was isolated using the NucliSens easyMAG platform (bioMérieux) for automated nucleic acid extraction.

Mutation analysis at codons 12 and 13 of the K-Ras gene, and exons 11 and 15 of the B-Raf gene was performed by direct sequencing of amplified PCR products. Genomic DNA was amplified by PCR using the following primers: FS 5′- TCA TTA TTT TTA TTA TAA GGC CTG CTG - 3′, RS 5′-CAA GAT TTA CCT CTA TTG TTG GAT CA-3′ (for codons 12 and 13 in exon 2 of K-Ras), BF11 5′-TCCCTCTCAGGCATAAGGTAA-3′, BR11 5′-TTATTGATGCGAACAGTGAATAT-3′ (for a glycine-rich loop region in exon 11 of the B-Raf gene), B2F 5′-TCATAATGCTTGCTCTGATAGGA–3′, B1R 5′-TAACTCAGCAGCATCTCAGG–3′ (for activation domain in exon 15 of the B-Raf gene). PCRs were performed in a total volume of 10 µl containing 2 µl of extracted genomic DNA, 1X PCR buffer, 1.5 mmol/L MgCl₂, 0.2 µmol/L of each primer, 0.1 mmol/L dNTPs and 1U of Taq DNA polymerase (EURx Ltd., Gdansk, Poland).

PCR conditions were as follows: 95°C for 10 min and 40 cycles of 95°C for 20 sec, 56°C for 30 sec (K-Ras and B-Raf in exon 11), 57°C for 30 sec (B-Raf in exon 15), 72°C for 30 sec, and finally 5 min at 72°C. Amplification products were purified using the DNA Gel-Out Kit (DNA GDANSK). Automated sequencing was performed using the Big Dye Terminator Cycle Sequencing kit version 3.1 (Applied Biosystems).

Sequencing reactions were purified using the ExTerminator Kit (DNA GDANSK), and analyzed on an ABI PRISM 377 DNA sequencer (Applied Biosystems). A wild-type control DNA sample (without K-Ras and B-Raf mutations) and a known mutation sample were also included in the experiment. The presence of a mutation was confirmed by sequencing at least two independent PCR products.

Detection of K-Ras mutations in codon 12 was performed by enriched non-radioactive single-step PCR-restriction fragment length polymorphism (RFLP) as described previously (Banerjee et al., 1997), with some modifications.

First-round PCR primers K1 5′-ACT GAA TAT AAA CTT GTG GTA GTT GGA CCT-3′ and DD5P 5′-TCA TGA AAA TGG TCA GAG AA-3′ were designed to create a restriction site for the restriction endonuclease BstOI (Promega) within the amplified product. The upstream primer K1 is immediately upstream of K-Ras codon 12 and introduces a G to C substitution at the first position of codon 11, creating a BstOI restriction site (5′-CCTGG-3′) in the amplified fragment. This site overlaps with the first 2 nucleotides of codon 12 and is lost when a codon 12 mutation is present. As a result, the restriction endonuclease BstOI recognizes the sequence 5′-CCTGG-3′ in K-Ras codon 12 wild-type PCR products and digests them, without affecting mutant PCR products.

Second-round PCR primers K1 and K2 5′-TCA AAG AAT GGT CCT GGA CC-3′ created another restriction site in the final segment of the PCR product, which served as an internal control for the restriction digestion. PCR products containing codon 12 mutations were mainly amplified in the second round, because wild-type products were digested in the previous step. These products will contain only one restriction site for BstOI near their 3′-end. Any non-digested PCR products containing wild-type codon 12 sequence that are amplified during the second PCR round will contain two BstOI restriction sites—one identical to that in the mutant molecules and the second overlapping with the codon 12 sequence (introduced by the K1 primer).

The products of the second PCR amplification were also digested with BstOI. The digestion products were electrophoresed on a 3% agarose gel and stained with ethidium bromide. Non-restricted PCR products were 157 bp, wild-type products were 113 bp and mutant codon 12 products were 142 bp in size. A normal control DNA sample (without the K-Ras codon 12 mutation) and a known mutation sample were included in all experiments. The results of PCR detection were verified by direct DNA sequencing.

The Chi-square test was used to investigate the differences between the 2 treatment groups with respect to baseline characteristics and response rates. Time to disease progression and overall survival (OS) were summarized as Kaplan-Meier estimates. The log-rank test was used in the Kaplan–Meier survival analyses to assess the effect of variables on time to disease progression and OS.

Multivariate analyses of time to disease progression and OS were performed by Cox proportional-hazard regression using the forward stepwise method; all variables found to be significant in the univariate analysis were included in the multivariate analysis. Statistical calculations were performed using STATISTICA for Windows Version 7.0 software.