Clinical Implications

KRAS and NRAS are closely related RAS oncogene family members, and mutations in either gene at codons 12, 13 (exon 2), codon 61 (exon 3) and codon 146 (exon 4) result in increased levels of guanosine triphosphate-bound RAS proteins. Overactive RAS signaling promotes oncogenesis. In colorectal carcinoma (CRC), KRAS and NRAS mutations at these codons are found in up to 50% of cases and predict a lack of response to anti-EGFR therapy. Most RAS mutations are point mutations occurring in KRAS exon 2 (codons 12 or 13; about 40%). Other RAS mutation are less frequent with the most common mutations occurring in KRAS exons 3 and 4 and NRAS exons 2 and 3.

The BRAF V600E mutation is found in about 10% of metastatic colorectal carcinoma and has been associated with the presence of microsatellite instability. Overall, patients with BRAF mutant CRC have low response rates to conventional therapies and adverse prognosis. While BRAF V600-mutated melanomas are sensitive to BRAF-inhibitors, BRAF V600-mutated CRCs may not be as sensitive. Activation of EGFR in colorectal cancer could explain why colorectal cancers generally have a lower response to BRAF inhibitors. Therefore, it is advised to initiate combination therapy with EGFR- and BRAF-inhibitors.

Methodologyl

Cobas® KRAS Mutation Test:

  • Manual specimen preparation to obtain genomic DNA from one or two 5 µm thick sections of FFPE CRC tissue containing at least 10% tumor cells.
  • PCR amplification of target DNA (exon 2 containing codons 12 and 13, exon 3 containing codon 61) using complementary primer pairs, two oligonucleotide probes labelled with fluorescent dye, and the Cobas z 480 analyzer.

KRAS Mutation Test v2 (LSR):

  • Manual sample preparation to obtain genomic DNA from FFPE tissue.
  • PCR amplification and detection of targeted KRAS sequences (range from 79 – 114 base pairs) using complementary primer pairs, oligonucleotide probes labeled with fluorescent dyes, and the Cobas z 480 analyzer.

The BRAF/NRAS Mutation Test (LSR) is based on two major processes:

  • Manual sample preparation to obtain genomic DNA from FFPE tissue.
  • PCR amplification and detection of targeted BRAF sequences (range from 101 – 120 base pairs)
    and NRAS sequences (range from 94 – 121 base pairs ) using complementary primer pairs,
    oligonucleotide probes labeled with fluorescent dyes, and the Cobas z 480 analyzer.

Specimen Requirements

Acceptable specimens for the assay are FFPE colorectal carcinoma tissue specimens with a fixation time of 6-48 hours.

Volume

One representative paraffin block is preferred. Alternatively, for resection specimens a minimum of 5 unstained tissue sections (5 µm thick) is required (full RAS testing).

Storage and Shipment Instructions

Maintain and ship specimens at ambient temperature.

Limitations

Insufficient tumor content may not allow the detection of KRAS/NRAS/BRAF mutations: 10% of tumor cells is required. Tumor content is evaluated prior to nalysis and macrodissection is performed.Fixatives other than formalin or prolonged fixation time may give rise to inadequate results.

Turn-Around Time

5 to 7 business days for respectively slides and paraffin blocks.