We assessed the prognostic value of KRAS codon 12 and codon 13 mutations in tumor tissue from patients with advanced CRC recruited into a phase III clinical trial using CAPOX or FUFOX treatment regimens. This is the first randomized phase III trial retrospectively investigating the role of codon 13 mutations in advanced CRC patients treated with oxaliplatin combination chemotherapy only, without the addition of a monoclonal antibody. While the overall response rate in codon 13 patients was significantly lower, PFS was not different in the three KRAS mutational groups. Interestingly, we found a substantial difference in PFS between patients with codon 12 and 13 mutant tumors when looking at infusional 5-FU versus capecitabine based regimens. Patients with codon 13 mutations seem to benefit more in terms of PFS from the oral capecitabine based protocols. Moreover, there was a strong trend towards better overall survival in patients with wild type KRAS compared to all mutant KRAS patients. Lastly, when analysing OS in patients who received second and further line therapy we found that KRAS wild type and KRAS mutant patients alike showed a significantly higher OS post progression when treated with cetuximab.
A number of studies have looked at the potential prognostic or predictive value of KRAS mutations on response rates and survival in patients with CRC and several studies found a negative impact of KRAS mutations on prognosis [3, 7, 15–17].
Recently, a number of randomized trials have included translational research programs to evaluate certain target genes and their role as prognostic or predictive markers in patients with metastatic disease. Thereby, the mutational status of KRAS has now been established as a strong predictive marker of resistance to anti-EGFR-antibody treatment [5, 8, 10], although some trials could not fully confirm these results [4, 11]. We still do not exactly know whether KRAS mutations influence the response to other treatment regimens such as standard chemotherapy or bevacizumab combinations. While bevacizumab efficacy seems independent from the KRAS status , the activity of certain chemotherapeutic agents may be influenced by KRAS mutations. There, patients seem to do worse under oxaliplatin combinations when carrying a mutant KRAS gene within their primary cancer [4, 5, 7, 16, 19, 20]. For example, in the recently reported COIN study patients under oxaliplatin/5-FU combinations showed a median PFS of 8.6 months in the wild type KRAS cohort while median PFS was only 6.9 months in the KRAS mutant cohort . Similarily, ORR was lower in KRAS mutant patients receiving chemotherapy with oxaliplatin only (41% vs. 50%). Furthermore, another recent small study evaluated the KRAS status in 66 patients receiving a second line chemotherapy with oxaliplatin and infusional 5-FU refractory to 5-FU/irinotecan based chemotherapy . This study found a significantly lower response rate (7% vs. 27%, p = 0.026) and significantly shorter median PFS (3.1 vs. 5.2 months, p = 0.007) for patients with mutant KRAS tumors compared to patients with wild type KRAS tumors under oxaliplatin containing therapy.
Very recently, experimental and some clinical reports suggested that not all KRAS mutations behave alike [12, 13, 21]. In fact, there is evidence that patients carrying mutations in codon 13 of the KRAS oncogene which is found in about 8% of patients with advanced CRC have a substantially worse overall prognosis but may, on the other hand, benefit from anti-EGFR-treatment.
In our study we found a similar rate of codon 13 mutations as described before. The overall response rate in patients with codon 13 mutations in our analysis was as low as 23%, significantly lower than in patients with wild type or codon 12 mutations. The CRYSTAL- and the OPUS-studies alike found low response rates in patients with codon 13 mutations treated with combination chemotherapy only (17% for irinotecan combinations and 33% for oxaliplatin combinations) . In contrast to previous reports, ORR was substantially higher in this codon 13 mutant patient cohort when cetuximab was added.
Our study was conducted using combination chemotherapy with oxaliplatin in first line treatment without the addition of monoclonal antibodies. Interestingly, the PFS and OS in our codon 13 cohort compared to the other mutated groups and other previously published works was rather long with a PFS of 10 months and an OS of 16.5 months . We do not know why PFS and OS of the codon 13 cohort in our study was prolonged when response rates were as low as 23%. Either low patient numbers or a yet unknown functional mechanism of codon 13 mutated KRAS proteins within colon cancers may be responsible for the observation.
The present analysis also suggests that there may be an interaction between the type of KRAS mutation and the mode of application of 5-FU, i.e. whether administered intravenously or orally as capecitabine. In particular, patients with codon 13 mutations showed longer median PFS intervals when receiving capecitabine compared to infusional 5-FU. Although the overall efficacy of infusional 5-FU and capecitabine in advanced CRC has been found to be comparable  there may be some patient subpopulations or treatment regimen where the mode of application of the fluoropyrimidine is more crucial. For example, anti-EGFR antibodies in wild type KRAS patients may only be active when infusional 5-FU regimens are used, but not when capecitabine based protocols are applied .
The potential resistance of KRAS mutated tumors to oxaliplatin containing regimens seems interesting and may reveal more general mechanisms of drug resistance in cancers. Oxaliplatin belongs to the platinum containing compounds like cisplatinum and carboplatinum. Metabolites of platinum compounds interact with DNA and form crosslinks. In addition, platinum-DNA-adducts strongly inhibit DNA polymerases and therefore act antineoplastic. Some authors have studied in cell culture systems and preclinical models the influence of oncogenic RAS mutations on the activity of platinum compounds and found that the nucleotide excision repair protein ERCC-1 may be upregulated through activated RAS. ERCC-1 may subsequently activate DNA repair capacity and thus mediate platinum resistance. A recent study evaluated the role of ERCC-1 mRNA levels in 191 patients treated with FOLFOX within the CONFIRM1 and CONFIRM2 studies. Low ERCC-1 gene expression was correlated with higher response rates to FOLFOX chemotherapy and better overall survival. In contrast patients with high ERCC-1 did not have benefit from FOLFOX chemotherapy . Of note, we have examined the expression of ERCC-1 by immunohistochemistry in our cohort and found no obvious correlation with KRAS status, response rates or survival and ERCC-1 expression (data not shown). The analysis of the ERCC-1 expression levels by RT-PCR as reported by other studies has not been performed so far .
Median overall survival after first progression correlated with cetuximab-treatment in patients bearing KRAS wild type and mutant tumors alike. The role of codon 13 mutation in this setting appears minor since there were only two patients with a codon 13 mutated tumor in the cohort with KRAS mutations of whom we know about cetuximab application. However, there are at least two limitations to our analysis. First, numbers are low in particular in the cetuximab group and secondly addition of cetuximab was not randomized for. There seems to be a selection of patients with good performance status and good prognosis who received third line therapy compared to those who did not. Therefore, we can not draw definite conclusions from our analysis whether anti-EGFR antibodies are effective in patients with KRAS G13D mutations or not.
We presently can not draw final conclusions regarding patient management from this study. It remains unclear whether chemotherapy backbones with irinotecan are less prone to interactions with mutated KRAS because data regarding this issue are conflicting. Independent validation of the findings is essential. Therefore, the standardized, thorough and comprehensive collection of tissue and blood samples of all trial patients within independent cancer tissue banks should be a major goal of modern clinical cancer trials.