KRAS gene mutation in a series of unselected colorectal carcinoma patients with prognostic morphological correlations: a pyrosequencing method improved by nested PCR.

INTRODUCTION: Inhibition of EGFR is a strategy for treating metastatic colorectal cancer (CRC) patients. KRAS sequencing is mandatory for selecting wild-type tumor patients who might benefit from this treatment. DNA from formalin-fixed paraffin-embedded (FFPE) tissues is commonly used for routine clinical detection of mutations, and its amplification succeeds only when all preanalytical histological processes have been controlled. In cases that are not properly processed, the DNA results can be poor, with low peak pyrosequencing findings. We designed and tested a pair of forward and reverse primers for a nested PCR method, followed by pyrosequencing, in a single Latin American institution series of 422 unselected CRC patients, correlating KRAS mutations with pathological and clinical data. MATERIALS AND METHODS: Patient DNA samples from tumors were obtained by scraping or laser microdissection of cells from FFPE tissue and extracted using a commercial kit. DNA was first amplified by PCR using 2 primers that we designed; then, nested PCR was performed with the amplicon from the preamplification PCR using the KRAS PyroMark™ Q96 V2.0 kit (Qiagen). Pathological data were retrieved from pathology reports. RESULTS: KRAS mutation was observed in 33% of 421 cases. Codon 12 was mutated in 76% of cases versus codon 13 in 24%. Right-sided CRCs harbored more KRAS mutations than left-sided tumors, as did tumors that presented with perineural invasion. CONCLUSION: Our findings in this Latin American population are consistent with the literature regarding the frequency of KRAS mutations in CRC, their distribution between codons 12 and 13, and type of nucleotide substitution. By combining nested PCR and pyrosequencing, we achieved a high rate of conclusive results in testing KRAS mutations in CRC samples - a method that can be used as an ancillary test for failed assays by conventional PCR.

KRAS insertions in colorectal cancer: what do we know about unusual KRAS mutations?

INTRODUCTION: KRAS mutations are negative predictors of the response to anti-EGFR therapy in colorectal carcinomas (CRCs). Point mutations in codons 12, 13, and 61 are the most common KRAS mutations in CRC. There are few reports on insertions in KRAS, and little is known about its ability to activate the RAS pathway. The scarcity of data regarding insertion frequencies and nucleotide additions in KRAS impedes the management of patients with such mutations. We present data on KRAS insertions in CRC and discuss a case. MATERIALS AND METHODS: Pyrosequencing and Sanger sequencing were performed to identify KRAS and BRAF mutations in paraffin-embedded samples of CRC. Expression of mismatch repair proteins was examined by immunohistochemistry. RESULTS: We detected a GGT insertion between codons 12 and 13 (c.36_37insGGT;p.G12_G13insG) in a CRC patient. We found that insertions in KRAS is very rare in CRC and that the most frequent type of insertion is c.36_37insGGT. CONCLUSIONS: KRAS gene insertions represent a diagnostic and clinical challenge due to the difficult and unusual pyrosequencing findings and the lack of information regarding its clinical impact.