Lack of evidence for KRAS oncogenic mutations in triple-negative breast cancer

Abstract

Background: Mutational analysis of the KRAS gene has recently been established as a complementary in vitro diagnostic tool for the identification of patients with colorectal cancer who will not benefit from anti-epidermal growth factor receptor (EGFR) therapies. Assessment of the mutation status of KRAS might also be of potential relevance in other EGFR-overexpressing tumors, such as those occurring in breast cancer. Although KRAS is mutated in only a minor fraction of breast tumors (5%), about 60% of the basal-like subtype express EGFR and, therefore could be targeted by EGFR inhibitors. We aimed to study the mutation frequency of KRAS in that subtype of breast tumors to provide a molecular basis for the evaluation of anti-EGFR therapies.

Methods: Total, genomic DNA was obtained from a group of 35 formalin-fixed paraffin-embedded, triple-negative breast tumor samples. Among these, 77.1% (27/35) were defined as basal-like by immunostaining specific for the established surrogate markers cytokeratin (CK) 5/6 and/or EGFR. KRAS mutational status was determined in the purified DNA samples by Real Time (RT)-PCR using primers specific for the detection of wild-type KRAS or the following seven oncogenic somatic mutations: Gly12Ala, Gly12Asp, Gly12Arg, Gly12Cys, Gly12Ser, Gly12Val and Gly13Asp.

Results: We found no evidence of KRAS oncogenic mutations in all analyzed tumors.

Conclusions: This study indicates that KRAS mutations are very infrequent in triple-negative breast tumors and that EGFR inhibitors may be of potential benefit in the treatment of basal-like breast tumors, which overexpress EGFR in about 60% of all cases.

Figure 1

Representative immunohistochemistry of a basal-like breast tumor showing negative staining for the hormone receptors (ER and PR) and HER2 and positive staining for EGFR and CK5/6.

Figure 2

Detection of KRAS mutations by RT-PCR. A, The graph shows a representative amplification curve (ΔRn vs cycle) from 100 ng of genomic DNA prepared from a triple-negative tumor sample. RT-PCR reactions were performed with primers specifically designed to amplify wild-type KRAS (red) or the following mutants: Gly12Ala (green), Gly12Asp (blue), Gly12Arg (yellow), Gly12Cys (Pink), Gly12Ser (brown), Gly12Val (purple), Gly13Asp (grey). Brown lines correspond to the amplification profile of an internal control included in each reaction to check for false positives. B, As a positive control, genomic DNA was obtained from a colon carcinoma biopsy and subjected to RT-PCR as in A. Note the presence of the Gly12Cys mutation.