Test name

NRAS Mutation by Sequencing (NRAS Mutation)

Purpose of the test

This is a clinical test intended for: Diagnosis, Predictive, Prognostic

Condition

4 conditions tested.

  • Cutaneous melanoma, lab preferred: Malignant melanoma
  • Carcinoma of colon (CRC)
  • Lung cancer
  • Papillary thyroid carcinoma

Methodology

  • Sequence analysis of select exons
  • Bi-directional Sanger Sequence Analysis
  • ABI 3130XL or 3730XL

Clinical utility

Guidance for selecting a drug therapy and/or dose

Testing strategy

Patients diagnosed with melanoma, colorectal, lung, and thyroid cancers will be tested for NRAS gene mutations. This assay will help to guide their therapy (such as consideration of treatment with EGFR antagonists cetuximab and panitumumab for patients with metastatic colorectal cancer, or consideration of treatment with TKIs for melanoma patients), as well as to get diagnostic/prognostic information (disease prognosis for melanoma patients and differential diagnosis of thyroid cancer patients).

Test services

Clinical Testing/Confirmation of Mutations Identified Previously

Target population

This test is appropriate for patients diagnosed with melanoma, colorectal, lung, and thyroid cancers.

Clinical utility

Guidance for selecting a drug therapy and/or dose

Methodology

Molecular Genetics

  • Sequence analysis of select exons
  • Bi-directional Sanger Sequence Analysis
  • ABI 3130XL or 3730XL

Test procedure

Exons 2 and 3 of the NRAS gene are PCR amplified using gene specific primers. The resulting DNA fragment is cleaned and sequenced bi-directionally using the BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies). The sequencing reaction is cleaned using a sephadex column and loaded into a ABI genetic analyzer. M13 forward and reverse primers are utilized for sequencing.

Confirmation of results

NRAS mutations identified in a single direction are confirmed in sequencing of the opposite DNA strand.

Analytical Validity

The accuracy of the NRAS bi-directional sequencing assay at Cancer Genetics, Inc. (CGI) was determined to be 95% by inter-lab assay. There was 100% concordance between the NRAS Sanger bi-directional sequencing assay results performed by two different operators on three different days. In addition, this assay was found to be precise as there was 100% concordance between assay results performed in triplicate within a single run by one operator. The sensitivity for accurate identification of the known alterations in the MT-NRAS cell line harboring mutations in exon 2 was greater than or equal to 2.5%. The sensitivity for accurate identification of the known alterations in the MT-NRAS cell line harboring mutations in exon 3 was greater than or equal to 5%. Additionally, the LOD of this assay can be reached as low as 1 ng MT DNA input . Finally, the Primer-BLAST results for NRAS PCR amplification primers demonstrated that the forward and reverse primer pairs for exons 2 and 3 were specific for the intended NRAS target and produced an amplicon of the expected length.

Assay Limitation(s)

Genomic DNA or DNA isolated from FFPE specimens at CGI was stored for no longer than 30 days at 2–8°C. DNA was placed in a -80°C freezer after 30 days for long-term storage. A minimum of 1 ng input DNA is required to detect 2.5% mutation against 97.5% wild-type genomic DNA at exon 2 of the NRAS gene. A minimum of 1 ng input DNA is required to detect 5% mutation againts 95% wild-type genomic DNA at exon 3 of the NRAS gene. All decalcified specimens are rejected.