NRAS TARGETED MUTATION ANALYSIS
Label Name: NRAS
Lab Discipline: Molecular Diagnostics
Institution:  Duke University Health System 
EAP ID:  LAB9173 
Last Review:  3/17/2017 10:05:47 AM
Specimen Type
  Tissue
Container & Volume
  Age Group   Container   Volume  
  0  - 18 Years CHECK WITH LABORATORY 1  ML
Collection Notes
  All:
  • Formalin Fixed Paraffin Embedded Tissue: The laboratory can receive either a paraffin embedded tissue block or four freshly cut (within one week)5uM thick unstained slides containing 3 to 20 square mm of tissue. Unstained slides should be accompanied by an H&E stained slide for histologic evaluation.


 
Transport
  Transport paraffin embedded tissue block or slides at ambient temperature.
Turn Around Time -  Routine: 14 days   Stat: N/A
Reference Values
NRAS
Mutation not detected
Methodology
  This assay uses purified genomic DNA that is extracted from macro-dissected formalin fixed paraffin-embedded tumor tissue blocks. Nine freshly cut (within one week) unstained slides should be submitted containing 5 mm2 to 20 mm2 of tissue. The slides should be prepared in the Molecular Diagnostics Lab following procedure MD-082 (Paraffin Microtomy), Ben Franklin Histology or Image Cytometry Lab -external clients may send unstained slides in lieu of a block to be cut in-house. An H&E stained slide should be prepared and submitted for reference purposes (unstained slide should be taken to Histology for staining). The pathologist on service will circle the tumor-containing area on the H&E slide and indicate approximate percentage of tumor cells present.

Forward and reverse primers, biotin-labeled, are used to amplify exons 2 and 3 (codons 12+13 and 61) of the NRAS gene. The biotinylated PCR product is captured by streptavidin-coated sepharose beads, denatured and separated in sodium hydroxide, and washed to produce a single-stranded DNA template. A sequencing primer is then hybridized to the PCR-amplified DNA template, and incubated with enzymes (DNA polymerase, ATP sulfurylase, luciferase and apyrase) and substrates (adenosine 5’ phosphosulfate and luciferin). Deoxyribonuckeotide triphophates (dNTP) are added to the reaction, one at a time, according to a predetermined dispensation order. The DNA polymerase incorporates the available nucleotide into the DNA strand if it is complementary to the base in the template strand. When a nucleotide is incorporated into the DNA strand, pyrophosphate(PPi) is released in a quantity equimolar to the number of nucleotides added. The released pyrophosphate is converted to ATP by adenosine 5’ phophosulfate, which is then used to catalyze the conversion of luciferin to oxyluciferin by luciferase, generating visible light in an amount proportional to the amount of ATP. The generated light is detected by a charge coupled device camera, and the signals used to generate a pyrogram® with peak heights proportional to the number of nucleotides incorporated into the DNA strand. Apyrase degrades any un-incorporated nucleotides and ATP following a dispensation, enabling the next nucleotide to be released and allowed to bind to the DNA strand. The Pyromark Vacuum Prep Workstation is used to prepared single-stranded biotin-labeled PCR product for pyrosequencing which is performed on the Qiagen Pyromark Q24 instrument.

This test was developed and its performance characteristics determined by the DUHS Clinical Molecular Diagnostics Laboratory. It has not been cleared or approved by the U.S. Food and Drug Administration. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement Amendments of 1988 ("CLIA") as qualified to perform high complexity clinical testing.


   
   
Clinical Significance and Interpretive Data
    The Neuroblastoma RAS viral oncogene homolog (NRAS) maps to chromosome 1p13.1 and encodes a cytoplasmic membrane bound GDP/GTP-binding protein from the RAS family. NRAS is activated by many proteins and pathways including growth factor receptor tyrosine kinases such as the EGF receptor and non-receptor tyrosine kinases such as ABL1. Activation of NRAS results in numerous signaling cascades that regulate cell proliferation, differentiation, and apoptosis. Under normal conditions, NRAS activation is short-lived and regulated by the hydrolysis of GTP. However, specific single amino acid substitutions in codons 12, 13 and 61 of NRAS generate a constitutively active GTP-bound protein. Constitutive activation of NRAS transforms the gene into a highly effective oncogene that plays a role in many human cancers, including colorectal adenocarcinoma, non-small cell lung carcinoma, and malignant melanoma.

REFERENCES:
McKusick, VA. Mendelian Inheritance in Man. A Catalog of Human Genes and Genetic Disorders. Baltimore: Johns Hopkins University Press, 1998 (12th edition). *164790

Di Fiore, F et al. Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated with cetuximab plus chemotherapy. British Journal of Cancer, 96:1166-1169, 2007.

De Rock, W et al. KRAS wild-type state predicts survival and is associated to early radiological response in metastatic colorectal cancer treated with cetuximab. Annals of Oncology 19(3):505-515, 2007

Lievre, A et al, KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab. J Clin Oncol. 26:374-379, 2008.

Amado, R et al. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer, J Clin Oncol. 26:1626-1634, 2008.

Arcila, M et al. Detection of KRAS and BRAF Mutations in Colorectal Carcinoma: Roles for High-Sensitivity Locked Nucleic Acid-PCR Sequencing and Broad Spectrum Mass Spectrometry Genotyping. The Journal of Molecular Diagnostics. 13(1):64-73, 2011.

Douillard, JY et al. Panitmumab-FOLFOX4 Treatment and RAS Mutations in Colorectal Cancer. N Engl J Med 369(11):1023-1034, 2013.

Vaughn, C et al. Frequency of KRAS, BRAF, and NRAS Mutations in Colorectal Cancer. Genes, Chromosomes & Cancer. 50:307-312, 2011.

De Roock, W et al. Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis. Lancet Oncol. 11:753-762, 2010.

Peeters, M et al. Massive parallel tumor multigene sequencing to evaluate response to Panitumumab in a randomized Phase III study of metatatic colorectal cancer. Clin Cancer Res. 19(7):1902-1912, 2013.

Ohashi, K et al. Characteristics of lung cancers harboring NRAS mutations. Clin Cancer Res. 19(9):2584-2591, 2013.

Carlino MS, et al. Correlation of BRAF and NRAS mutation status with outcome, site of distant metastasis and response to chemotherapy in metastatic melanoma. Br J Cancer 2014;111:292-299.

Chapman PB, Hauschild A, Robert C et al. Improved Survival with Vemurafenib in Melanoma with BRAF V600E Mutation. N Engl J Med 2011; 364(26):2507-16.

Ellerhorst JA, Greene VR, Ekmekcioglu S et al. Clinical correlates of NRAS and BRAF mutations in primary human melanoma. Clin Cancer Res 2011; 17:229–35.

Joseph EW, Partilas CA, Poulikakos PI et al. The RAF inhibitor PLX4032 inhibits ERK signaling and tumor cell proliferative in a V600E BRAF-selective manner. Proc Natl Acad Sci USA; 2010; 107:14903-8.
NCCN Clinical Practice Guidelines in Oncology. Melanoma, Version 1.2015.

NCCN Clinical Practice Guidelines in Oncology. Colon Cancer, Version 3.2014.

Ohashi, K et al. Characteristics of lung cancers harboring NRAS mutations. Clin Cancer Res. 19(9):2584-2591, 2013.

Peeters M, et al. Mutant KRAS codon 12 and 13 alleles in patients with metastatic colorectal cancer: assessment as prognostic and predictive biomarkers of response to panitumumab. J Clin Oncol. 2013;31(6):759-65.

Vaughn C, et al. Frequency of KRAS, BRAF, and NRAS mutations in colorectal cancer. Genes, Chromosomes & Cancer. 2011;50:307-312.

For additional information, please refer to the package inserts for Erbitux (cetuximab) and Vectibix (panitumumab).

QIAGEN. therascreen® NRAS Pyro® Kit Handbook. August 2011 (version 1)

   
Indications
    Approximately 2-5% of CRC tumors and <1% of lung tumors contain a mutation in the NRAS gene. NRAS mutations of codons 12, 13, and 61 represent approximately one-third, between six to eight percent, and over half of the NRAS mutation spectrum in CRC, respectively. There have been similar mutation frequencies reported in lung cancer. (Note: screening for NRAS mutations is not yet standard for KRAS negative tumors, and the NRAS mutation frequency may be under represented, in addition, the majority of studies have only examined KRAS non-mutated samples).
Studies have consistently demonstrated that cetuximab and panitumumab have little to no efficacy in patients with tumors that harbor activating KRAS mutations. However, patients with non-mutated KRAS tumors had a response rate between 17 and 41%. There has been an increase of studies screening for other mutations prevalent in lung and colorectal cancers: the lack of response in the KRAS non-mutated population has been found to be associated with mutations in genes in the EGFR pathway. In patients receiving panitumumab or cetuximab, the median overall survival, progression-free survival, and tumor size reduction was significantly better in patients with NRAS non-mutated statues when compared to patients with a RAS mutation.
Testing patient’s tumors for KRAS exon 2 (codon 12 and 13) has become standard practice, prior to the initiation of cetuximab or panitumumab therapy. Continued screening of KRAS non-mutated tumors for other mutations has become more prevalent. The result of a NRAS mutation can be used to predict a near complete lack of clinical benefit of cetuximab and panitumumab in mCRC patients.

NRAS mutations occur in approximately 15-25% of melanomas, and are mutually exclusive with BRAF mutations, which occur in approximately 40-50% of melanomas. NRAS mutations commonly occur in codons 12, 13, and 61, and result in activation of the RAS-RAF-MEK-MAPK pathway. For patients with metastatic melanoma, the NCCN recommends mutational analysis for consideration of treatment or clinical trials. There is currently no direct anti-NRAS agent, but trials of MEK inhibitors (and other inhibitors of the pathway) are ongoing. It is uncertain if there is any prognostic significance of NRAS mutations, as results to date have been conflicting. This test is intended for use as an aid in making individualized patient treatment decisions and is not a substitute for a physician's judgment and clinical experience.


   
Limitations
    • This assay may not detect an acquired mutation which is present below the 10% detection limit (ie, mutant cell population of 20%).
• Only exons 2 and 3 of the NRAS gene are examined. Specifically, this assay detects mutations in codons 12, 13, and 61.Variations outside of these regions will not be detected.
• The presence of a mutant population containing a large deletion, duplication, insertion, aberrant splicing, or sequence alteration adversely affecting primer binding may not be identified using these methods.
• Mutations or polymorphisms in the DNA oligonucleotide primer binding regions, poor DNA quality, insufficient DNA quantity or the presence of PCR inhibitors can result in uninterpretable, or rarely inaccurate, results.
• This procedure cannot detect duplication of NRAS.

   
Related Tests
    KRAS TARGETED MUTATION ANALYSIS