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Background: The progression of colorectal cancer (CRC) involves recurrent amplifications/mutations in the epidermal growth factor receptor (EGFR) and downstream signal transducers of the Ras pathway, KRAS and BRAF. Whether genetic events predicted to result in increased and constitutive signaling indeed lead to enhanced biological activity is often unclear and, due to technical challenges, unexplored. Here, we investigated proliferative signaling in CRC using a highly sensitive method for protein detection. The aim of the study was to determine whether multiple changes in proliferative signaling in CRC could be combined and exploited as a "complex biomarker" for diagnostic purposes., Methods: We used robotized capillary isoelectric focusing as well as conventional immunoblotting for the comprehensive analysis of epidermal growth factor receptor signaling pathways converging on extracellular regulated kinase 1/2 (ERK1/2), AKT, phospholipase Cγ1 (PLCγ1) and c-SRC in normal mucosa compared with CRC stage II and IV. Computational analyses were used to test different activity patterns for the analyzed signal transducers., Results: Signaling pathways implicated in cell proliferation were differently dysregulated in CRC and, unexpectedly, several were downregulated in disease. Thus, levels of activated ERK1 (pERK1), but not pERK2, decreased in stage II and IV while total ERK1/2 expression remained unaffected. In addition, c-SRC expression was lower in CRC compared with normal tissues and phosphorylation on the activating residue Y418 was not detected. In contrast, PLCγ1 and AKT expression levels were elevated in disease. Immunoblotting of the different signal transducers, run in parallel to capillary isoelectric focusing, showed higher variability and lower sensitivity and resolution. Computational analyses showed that, while individual signaling changes lacked predictive power, using the combination of changes in three signaling components to create a "complex biomarker" allowed with very high accuracy, the correct diagnosis of tissues as either normal or cancerous., Conclusions: We present techniques that allow rapid and sensitive determination of cancer signaling that can be used to differentiate colorectal cancer from normal tissue.

Background: Podocalyxin-like 1 (PODXL) is an anti-adhesive transmembrane protein that has been demonstrated to be an independent factor of poor prognosis in colorectal cancer (CRC). The gene encoding PODXL is located to chromosome 7, which also harbours the gene for the epidermal growth factor receptor (EGFR). The aim of this study was to examine the associations between PODXL and EGFR expression in CRC in vitro and in vivo., Methods: EGFR expression was analysed in tumours from three independent patient cohorts; cohort 1 (n = 533), cohort 2 (n = 259) and cohort 3 (n = 310), previously analysed for immunohistochemical PODXL expression and KRAS and BRAF mutations (cohort 1 and 3). Levels of EGFR and PODXL were determined by western blot in six different CRC cell lines., Results: High expression of PODXL was significantly associated with high EGFR expression (p < 0.001) in all three cohorts, and with BRAF mutation (p < 0.001) in cohort 1 and 3. High EGFR expression correlated with BRAF mutation (p < 0.001) in cohort 1. High EGFR expression was associated with adverse clinicopathological factors and independently predicted a reduced 5-year overall survival (OS) in cohort 1 (HR 1.77; 95 % CI 1.27-2.46), cohort 2 (HR 1.58; 95 % CI 1.05-2.38) and cohort 3 (HR 1.83; 95 % CI 1.19-2.81). The highest risk of death within 5 years was observed in patients with tumours displaying high expression of both EGFR and PODXL in cohort 1 and 3 (HR 1.97; 95 % CI 1.18-3.28 and HR 3.56; 95 % CI 1.75-7.22, respectively). Western blot analysis showed a uniform expression of PODXL and EGFR in all six examined CRC cell lines., Conclusions: The results from this study demonstrate that high expression of EGFR is an independent factor of poor prognosis in CRC. Moreover, strong links have been uncovered between expression of the recently proposed biomarker candidate PODXL with EGFR expression in CRC in vivo and in vitro, and with BRAF mutation in vivo. High expression of both PODXL and EGFR may also have a synergistic adverse effect on survival. These findings suggest a potential functional link in CRC between PODXL, EGFR and BRAF, all originating from chromosome 7, which may be highly relevant in the clinical setting and therefore merit future in-depth study.

Background: Molecular alterations are well studied in colon cancer, however there is still need for an improved understanding of their prognostic impact. This study aims to characterize colon cancer with regard to KRAS, BRAF, and PIK3CA mutations, microsatellite instability (MSI), and average DNA copy number, in connection with tumour dissemination and recurrence in patients with colon cancer., Methods: Disease stage II-IV colon cancer patients (n = 121) were selected. KRAS, BRAF, and PIK3CA mutation status was assessed by pyrosequencing and MSI was determined by analysis of mononucleotide repeat markers. Genome-wide average DNA copy number and allelic imbalance was evaluated by SNP array analysis., Results: Patients with mutated KRAS were more likely to experience disease dissemination (OR 2.75; 95% CI 1.28-6.04), whereas the opposite was observed for patients with BRAF mutation (OR 0.34; 95% 0.14-0.81) or MSI (OR 0.24; 95% 0.09-0.64). Also in the subset of patients with stage II-III disease, both MSI (OR 0.29; 95% 0.10-0.86) and BRAF mutation (OR 0.32; 95% 0.16-0.91) were related to lower risk of distant recurrence. However, average DNA copy number and PIK3CA mutations were not associated with disease dissemination., Conclusions: The present study revealed that tumour dissemination is less likely to occur in colon cancer patients with MSI and BRAF mutation, whereas the presence of a KRAS mutation increases the likelihood of disseminated disease.

Background: The clinical behaviour of colon cancer is heterogeneous. Five-year overall survival is 50-65% with all stages included. Recurring somatic chromosomal alterations have been identified and some have shown potential as markers for dissemination of the tumour, which is responsible for most colon cancer deaths. We investigated 115 selected stage II-IV primary colon cancers for associations between chromosomal alterations and tumour dissemination., Methods: Follow-up was at least 5 years for stage II-III patients without distant recurrence. Affymetrix SNP 6.0 microarrays and allele-specific copy number analysis were used to identify chromosomal alterations. Fisher's exact test was used to associate alterations with tumour dissemination, detected at diagnosis (stage IV) or later as recurrent disease (stage II-III)., Results: Loss of 1p36.11-21 was associated with tumour dissemination in microsatellite stable tumours of stage II-IV (odds ratio = 5.5). It was enriched to a similar extent in tumours with distant recurrence within stage II and stage III subgroups, and may therefore be used as a prognostic marker at diagnosis. Loss of 1p36.11-21 relative to average copy number of the genome showed similar prognostic value compared to absolute loss of copies. Therefore, the use of relative loss as a prognostic marker would benefit more patients by applying also to hyperploid cancer genomes. The association with tumour dissemination was supported by independent data from the The Cancer Genome Atlas., Conclusion: Deletions on 1p36 may be used to guide adjuvant treatment decisions in microsatellite stable colon cancer of stages II and III.

Background: Podocalyxin-like 1 (PODXL) is a cell-adhesion glycoprotein and stem cell marker that has been associated with an aggressive tumour phenotype and adverse outcome in several cancer types. We recently demonstrated that overexpression of PODXL is an independent factor of poor prognosis in colorectal cancer (CRC). The aim of this study was to validate these results in two additional independent patient cohorts and to examine the correlation between PODXL mRNA and protein levels in a subset of tumours., Method: PODXL protein expression was analyzed by immunohistochemistry in tissue microarrays with tumour samples from a consecutive, retrospective cohort of 270 CRC patients (cohort 1) and a prospective cohort of 337 CRC patients (cohort 2). The expression of PODXL mRNA was measured by real-time quantitative PCR in a subgroup of 62 patients from cohort 2. Spearman's Rho and Chi-Square tests were used for analysis of correlations between PODXL expression and clinicopathological parameters. Kaplan Meier analysis and Cox proportional hazards modelling were applied to assess the relationship between PODXL expression and time to recurrence (TTR), disease free survival (DFS) and overall survival (OS)., Results: High PODXL protein expression was significantly associated with unfavourable clinicopathological characteristics in both cohorts. In cohort 1, high PODXL expression was associated with a significantly shorter 5-year OS in both univariable (HR = 2.28; 95% CI 1.43-3.63, p = 0.001) and multivariable analysis (HR = 2.07; 95% CI 1.25-3.43, p = 0.005). In cohort 2, high PODXL expression was associated with a shorter TTR (HR = 2.93; 95% CI 1.26-6.82, p = 0.013) and DFS (HR = 2.44; 95% CI 1.32-4.54, p = 0.005), remaining significant in multivariable analysis, HR = 2.50; 95% CI 1.05-5.96, p = 0.038 for TTR and HR = 2.11; 95% CI 1.13-3.94, p = 0.019 for DFS.No significant correlation could be found between mRNA levels and protein expression of PODXL and there was no association between mRNA levels and clinicopathological parameters or survival., Conclusions: Here, we have validated the previously demonstrated association between immunohistochemical expression of PODXL and poor prognosis in CRC in two additional independent patient cohorts. The results further underline the potential utility of PODXL as a biomarker for more precise prognostication and treatment stratification of CRC patients.

Background: In cancer research the selection and definitions of survival endpoints are important and yet they are not used consistently. The aim of this study was to compare different survival endpoints in patients with primary colorectal cancer (CRC) and to understand the effect of second primary other cancer on disease-free survival (DFS) calculations., Methods: A population-based cohort of 415 patients with CRC, 332 of whom were treated with curative intention between the years 2000-2003, was analysed. Events such as locoregional recurrence, distant metastases, second primary cancers, death, cause of death and loss to follow-up were recorded. Different survival endpoints, including DFS, overall survival, cancer-specific survival, relapse-free survival, time to treatment failure and time to recurrence were compared and DFS was calculated with and without inclusion of second primary other cancers., Results: The events that occurred most often in patients treated with curative intention were non-cancer-related death (n = 74), distant metastases (n = 66) and death from CRC (n = 59). DFS was the survival endpoint with most events (n = 170) followed by overall survival (n = 144) and relapse-free survival (n = 139). Fewer events were seen for time to treatment failure (n = 80), time to recurrence (n = 68) and cancer-specific survival (n = 59). Second primary other cancer occurred in 26 patients and its inclusion as an event in DFS calculations had a detrimental effect on the survival. The DFS for patients with stage I-III disease was 62% after 5 years if second primary other cancer was not included as an event, compared with 58% if it was. However, the difference was larger for stage II (68 vs 60%) than for stage III (49 vs 47%)., Conclusions: The inclusion of second primary other cancer as an endpoint in DFS analyses significantly alters the DFS for patients with CRC. Researchers and journals must clearly define survival endpoints in all trial protocols and published manuscripts.

Background: Epidermal growth factor receptor inhibitor therapy is now approved for treatment of metastatic colorectal carcinomas (CRC) in patients with tumors lacking KRAS mutations. Several procedures to detect KRAS mutations have been developed. However, the analytical sensitivity and specificity of these assays on routine clinical samples are not yet fully characterised., Methods: The practical aspects and clinical applicability of a KRAS-assay based on Pyrosequencing were evaluated in a series of 314 consecutive CRC cases submitted for diagnostic KRAS analysis. The performance of Pyrosequencing compared to allele-specific, real-time PCR was then explored by a direct comparison of CE-IVD-marked versions of Pyrosequencing and TheraScreen (DxS) KRAS assays for a consecutive subset (n = 100) of the 314 clinical CRC samples., Results: Using Pyrosequencing, 39% of the 314 CRC samples were found KRAS-mutated and several of the mutations (8%) were located in codon 61. To explore the analytical sensitivity of the Pyrosequencing assay, mutated patient DNA was serially diluted with wild-type patient DNA. Dilutions corresponding to 1.25-2.5% tumor cells still revealed detectable mutation signals. In clinical practice, our algorithm for KRAS analysis includes a reanalysis of samples with low tumor cell content (< 10%, n = 56) using an independent assay (allele-specific PCR, DxS). All mutations identified by Pyrosequencing were then confirmed and, in addition, one more mutated sample was identified in this subset of 56 samples. Finally, a direct comparison of the two technologies was done by re-analysis of a subset (n = 100) of the clinical samples using CE-IVD-marked versions of Pyrosequencing and TheraScreen KRAS assays in a single blinded fashion. The number of samples for which the KRAS codon 12/13 mutation status could be defined using the Pyrosequencing or the TheraScreen assay was 94 and 91, respectively, and both assays detected the same number of codon 12 and 13 mutations., Conclusions: KRAS mutation detection using Pyrosequencing was evaluated on a consecutive set of clinical CRC samples. Pyrosequencing provided sufficient analytical sensitivity and specificity to assess the mutation status in routine formalin-fixed CRC samples, even in tissues with a low tumor cell content.

Background: There is an insufficient number of reliable prognostic and response predictive biomarkers in colorectal cancer (CRC) management. In a previous study, we found that high tumour tissue expression of tumour-associated trypsin inhibitor (TATI) correlated with liver metastasis and an impaired prognosis in CRC. The aim of this study was to investigate the prognostic validity of serum TATI (s-TATI) in CRC. We further assessed the prognostic value of carcino-embryonic antigen in serum (s-CEA) and the interrelationship between s-TATI and TATI in tissue (t-TATI)., Methods: Using an immunofluorometric assay, s-TATI levels were analysed in 334 preoperatively collected serum samples from patients with CRC. Spearman's Rho and Chi-square test were used for analysis of correlations between s-TATI and clinicopathological parameters, s-CEA and t-TATI. Kaplan-Meier analysis and Cox uni- and multivariate regression analysis were used to estimate disease free survival (DFS) and overall survival (OS) according to quartiles of s-TATI and cut-offs derived from ROC-analysis of s-TATI and s-CEA., Results: Increased levels of s-TATI were associated with a reduced DFS (HR = 2.00; 95% CI 1.40-2.84, P < 0.001) and OS (HR = 2.40; 95% CI 1.74-3.33, P < 0.001). (HR = 2.89; 95% CI 1.96-4.25). This association remained significant in multivariate analysis. The association for OS remained significant in multivariate analysis (HR = 1.51; 95% CI 1.03-2.22, P = 0.034 for DFS and HR = 1.78; 95% CI 1.25-2.53, P = 0.001 for OS). There was no significant association between s-TATI and t-TATI. The prognostic value of s-CEA was also evident, but somewhat weaker than for s-TATI., Conclusions: High preoperative s-TATI levels predict a poor prognosis in patients with CRC, and the prognostic value is independent of established prognostic parameters and t-TATI expression. These data suggest that s-TATI might be a useful marker for prognostic stratification in CRC.