KRAS and NRAS Mutation Testing (Colorectal Cancer)

1. 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).

2. KRAS Mutation Test v2 (LSR)

The KRAS Mutation Test v2 (LSR) is an allele-specific, real-time PCR test for the qualitative detection and identification of exon 2, 3, and 4 mutations in the V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) gene from formalin-fixed, paraffin-embedded tissue (FFPET). The test is designed to detect 28 unique mutations. The presence of mutations is detected with an analytical specificity of at least 99% and a detection limit of at least 1% mutant level in a background of wild-type genomic DNA.

The KRAS Mutation Test v2 (LSR) is based on two processes: (1) manual sample preparation to obtain genomic DNA from FFPET; and (2) PCR amplification and detection of target DNA using complementary primer pairs and oligonucleotide probes labeled with fluorescent dyes.

The KRAS Mutation Test v2 (LSR) uses primers that define specific base-pair sequences for each of the targeted mutations. Amplification occurs only in the regions of the KRAS gene between the primers; the entire gene is not amplified. The targeted KRAS sequences range from 79 – 114 base pairs. Mutation detection is achieved through PCR analysis with the Cobas z 480 analyzer. (1)

3. BRAF/NRAS Mutation Test (LSR)

The BRAF/NRAS Mutation Test (LSR) is an allele-specific, real-time PCR test for the qualitative detection and identification of exon 11 and 15 mutations in the proto-oncogene B-Raf (BRAF) gene and exon 2, 3, and 4 mutations in the neuroblastoma RAS viral oncogene homolog (NRAS) gene from formalin-fixed, paraffin-embedded tissue (FFPET).

The test is designed to detect 36 unique 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 BRAF/NRAS Mutation Test (LSR) is based on two major processes: (1) manual sample preparation to obtain genomic DNA from FFPET; and (2) PCR amplification and detection of target DNA using complementary primer pairs and oligonucleotide probes labeled with fluorescent dyes.

The BRAF/NRAS Mutation Test (LSR) uses primers that define specific base-pair sequences for each of the targeted mutations. Amplification occurs only in the regions of the BRAF or NRAS genes between the primers; the entire gene is not amplified. BRAF sequences range from 101 – 120 base pairs. NRAS sequences range from 94 – 121 base pairs. Mutation detection is achieved through PCR analysis with the Cobas z 480 analyzer. (2)

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 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 (3).

About 50% of melanomas harbor activating mutations in BRAF. Over 90% of BRAF mutations result in a nucleotide 1799 T>A change leading to a valine-to-glutamic acid substitution at position 600 (V600E). The BRAF V600E mutation causes uncontrolled signaling of the MAPK pathway leading to excessive cell growth and proliferation. Agents targeting activated mutant BRAF (BRAF-inhibitors) have proven to be successful in patients with metastatic melanoma harboring this mutation (1-3).

Apart from its predictive value in melanoma, the BRAF V600E mutation also has prognostic value in colorectal cancer and in lung cancer (NSCLC) (4,5,7). The BRAF V600E mutation is found in about 10% of metastatic colorectal carcinoma and has been associated with the presence of microsatellite instability (6). 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.

The BRAF V600E mutation is also found in 3-5% of all lung cancers (7). While BRAF-inhibitors have proven to be successful in V600E mutation-positive NSCLC patients, the FDA has approved use of the combination therapy with BRAF- and MEK-inhibitor for NSCLC patients with a V600E mutation based on the results of an international, multicenter, three-cohort, non-randomized, non-comparative, open-label, trial in patients with locally confirmed BRAF V600E mutation-positive metastatic NSCLC.

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/NRAS/BRAF mutations: 10% of tumor cells is required. 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

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

References

  1. Li et al. A Highly Verified Assay for KRAS Mutation Detection in Tissue and Plasma of Lung, Colorectal, and Pancreatic Cancer. Arch Pathol Lab Med. 2019 Feb;143:183-9
  2. Patten et al. A sensitive and accurate test for simultaneous detection of 36 BRAF and NRAS mutations. Journal of Clinical Oncology 2018 36:15
  3. Douillard JY et al. Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer. N Engl J Med. 2013 Sep 12;369(11):1023-34.

Updated on 03 December 2019