KRAS and NRAS Mutation Testing (Colorectal Cancer)

KRAS exon 2 (codons 12/13) and exon 3 (codon 61) mutation testing: Cobas® KRAS Mutation Test for In Vitro Diagnostic use (Roche).

The Cobas® KRAS Mutation Test is a real-time PCR test for the qualitative detection of somatic mutations in exon 2 (codons 12/13) and exon 3 (codon 61) of the KRAS gene using a DNA input of 100 ng. The test can detect 19 KRAS mutations. The presence of mutations is detected with an analytical specificity of at least 99% and a detection limit of at least 5% mutant level in a background of wild-type genomic DNA.

The Cobas® KRAS Mutation Test is based on two major processes: (1) 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; (2) PCR amplification of target DNA using complementary primer pairs and two oligonucleotide probes labelled with fluorescent dye. The primer pairs used define an 85 base-pair sequence for exon 2 containing KRAS codons 12 and 13, and a 75 base-pair sequence for exon 3 containing KRAS codon 61 in human genomic DNA. One probe is designed to detect the KRAS codon 12/13 sequence in exon 2, and the other probe is designed to detect the KRAS codon 61 sequence in exon 3 of the KRAS gene. Mutation detection is achieved by melting curve analysis by the cobas z 480 analyzer (1).

KRAS exon 4 (codon 146) mutation testing and NRAS exon 2 (codons 12/13) and exon 3 (codon 61) mutation testing: high resolution melting analysis (Laboratory Developed Test).

In case no mutations are detected in KRAS exons 2/3 using the Cobas® KRAS Mutation Test, the DNA extract is subjected to high resolution melting analysis on the Cobas z480 instrument (UDF mode) using the LightCycler 480 High Resolution Melting Master mix (Roche). 

A DNA input of 200 ng is required. The presence of mutations is detected with an analytical specificity of at least 99% and a detection limit of at least 5% mutant level (KRAS exon 4 and NRAS exon 2 assays) or 13% mutant level (NRAS exon 3 assay) in a background of wild-type genomic DNA.

Clinical Implication

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 signalling 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 (2). 

Specimen Requirements

Acceptable specimens for the assay are formalin-fixed, paraffin-embedded colorectal carcinoma tissue specimens with a fixation time of 6-48 hours.

Volume

1 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 mutations: 10% of tumor cells is required for KRAS exon 2/3/4 and NRAS exon 2 mutation analysis; > 25% of tumor cells is required for NRAS exon 3 mutation analysis. Tumor content is evaluated prior to analysis and macrodissection is performed. Fixatives other than formalin or prolonged fixation time may give rise to inadequate results.

Special requirements

None.

Turn-around-time

Five to 7 business days for respectively slides and paraffin blocks. In 2014, for KRAS exon 2/3 mutation analysis, the mean turn-around-time was 3 business days (mainly paraffin blocks).

References

  1. Harlé et al. Comparison of Cobas 4800 KRAS, TaqMan PCR and High Resolution Melting PCR assays for the detection of KRAS somatic mutations in formalin-fixed, paraffin-embedded colorectal carcinomas. Virchows Arch 2013;462(3):329-35.
  2. Douillard JY et al. Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer. N Engl J Med. 2013 Sep 12;369(11):1023-34.