ORIGINAL ARTICLE
Frequency and type of KRAS mutations in routine diagnostic analysis of metastatic colorectal cancer

https://doi.org/10.1016/j.prp.2009.07.010Get rights and content

Abstract

Mutation analysis of the KRAS oncogene is now established as a predictive biomarker in colorectal cancer (CRC). Large prospective clinical trials have shown that only CRCs with wild-type KRAS respond to anti-epidermal growth factor receptor (EGFR) treatment. Therefore, mutation analysis is mandatory before treatment, and reliable benchmarks for the frequency and types of KRAS mutations have to be established for routinely testing large numbers of metastatic CRCs.

A thousand and eighteen cases (879 primary tumors and 139 metastases) of metastatic colorectal cancer were analyzed for the KRAS mutational status of codons 12 and 13 of the KRAS gene by genomic sequencing in a routine setting. Results were analyzed separately for specimens derived from primary tumors and metastases.

KRAS mutations in codons 12 and 13 were present in 39.3% of all analyzed CRCs. The most frequent types of mutations were glycine to aspartate on codon 12 (p.G12D, 36.0%), glycine to valine on codon 12 (pG12V, 21.8%), and glycine to aspartate on codon 13 (p.G13D, 18.8%). They account for 76.6% of all mutations and prevail in primary tumors and distant metastases, indicating a robustness of the KRAS mutational status during neoplastic dissemination.

The frequency of KRAS mutations and the preponderance of three types of mutations in codons 12 and 13 in a large, unselected cohort of metastatic CRC confirm the previous data of small and selected CRC samples. Thus, a mutation frequency of 40% and a cluster of three mutation types (p.G12D, pG12V, and p.G13D) in primaries and metastases can be defined as benchmarks for routine KRAS analyses.

Introduction

Anti-EGFR-targeted therapies with monoclonal antibodies such as cetuximab (Erbitux®) or panitumumab (Vectibix®) are a successful strategy for the treatment of metastatic CRC in addition to or after failure of conventional chemotherapy. However, the EGFR-targeted therapies accomplish the partial response or stabilization of disease only in a subgroup of patients [1], [10], [11], [13], [15], [23], [25], [27]. Prospective clinical trials have clearly shown that only CRCs with wild-type KRAS proto-oncogene, a small G-protein downstream in the EGFR signaling cascade, respond to anti-EGFR treatment, whereas no response is observed in CRC with KRAS mutation. Consequently, the European Medicines Agency (EMEA) approved panitumumab (Vectibix®) and cetuximab (Erbitux®) only for the treatment of metastatic CRC with intratumorous wild-type KRAS status.

Activating mutations of the KRAS gene, resulting in EGFR-independent activation of the mitogen-activated protein kinase pathway (MAPK), have been reported in 30–54% of metastatic colorectal tumors. The most frequent alterations are detected in codon 12 (approximately 82% of all reported KRAS mutations) and codon 13 (approximately 17%). Mutations in other positions, such as codons 61 and 146, have also been reported. However, these alterations account for a minor proportion (1–4%) of KRAS mutations, and their clinical relevance in CRC still remains unclear [2], [3], [4], [5], [7], [8], [9], [12], [16], [17], [18], [19], [20], [26], [30], [31].

Until now, the overall frequency of KRAS mutations has been estimated according to the results of clinical studies, drug admission trials, or retrospective multi-center studies with different inclusion criteria and variable ethnical backgrounds, whereas large cohorts of unselected metastatic CRC with a demand for routine EGFR-targeted therapy have not been analyzed. Meanwhile, large numbers of metastatic CRCs (UICC Stage IV) have to be routinely tested for KRAS mutations. Therefore, reliable benchmarks for the frequency and types of KRAS mutations are needed for the implementation and quality assurance of KRAS mutation analysis of metastatic CRC in routine diagnosis. Moreover, primary tumors and metastases have to be compared to evaluate the robustness or variation of the KRAS mutation status during neoplastic dissemination.

In conclusion, our study is the first to provide data on the frequency and types of KRAS mutations from a large homogeneous Caucasian cohort of unselected metastatic CRC tested in a routine setting.

Section snippets

Tissue selection

At the Department of Pathology, Ludwig-Maximilians Universität Müchen, in the period from January to November 2008, we analyzed the KRAS mutation status in 1018 unselected cases of metastatic CRC (UICC Stage IV), for which anti-EGFR-therapy was intended. The mean age at the date of receipt of the patients' specimens was 63.8 years (±10.87). 62.4% of the patients were male, and 37.6% were female. For all analyses, formalin-fixed and paraffin-embedded tissue specimens were used. Four hundred and

Results

Of all cases investigated, 39.3% showed mutations in exon 2 codons 12 and 13 of the KRAS proto-oncogene. Conversely, 60.7% showed a wild-type (WT) sequence (Fig. 1). Three patients presented two mutations in codon 12. The most common mutations were glycine to aspartate on codon 12 (p.G12D, 36.0% of mutated tumors; 144 of 1018), glycine to valine on codon 12 (pG12V, 21.8% of mutated tumors; 87 of 1018), and glycine to aspartate on codon 13 (p.G13D, 18.8% of mutated tumors; 75 of 1018). These

Discussion

We present the results of routine KRAS mutation analyses in a large cohort of unselected metastatic CRCs (UICC Stage IV) showing a mutation frequency of 39.3%. This confirms the data of previous studies of selected cohorts, in which the frequency ranges from 30% to 54%. Up to now, the estimates of the mutation frequency of KRAS for metastatic CRCs have been based on selective clinical studies or drug admission trials with variable inclusion criteria. Most of the studies have evaluated smaller

Competing interests

Thomas Kirchner and Andreas Jung received financial support from AMGEN GmbH Germany and Merck-Serono KG for the implementation of a quality assurance system in Germany for the routine molecular-pathological analysis of KRAS mutations, which is under the auspices of the QuIP (Quality Initiative in Pathology) structure. QuIP is a structure founded and supported by the Deutsche Gesellschaft für Pathologie (German Society for Pathology) and the Bundesverband Deutscher Pathologen (Federation of the

Acknowledgments

We thank Mrs. Gaby Charell, Jutta Hügel-Tegge, Nicole Perera, Irina Redich, and Karina Windhorst for their excellent technical assistance.

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