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Test ID: NRAS
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NRAS Mutation Analysis.
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NRAS mutation detection by Real Time - PCR
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Useful For
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NRAS mutations serves as prediction biomarkers for treatment responses and prognosis in several cancer types, including colorectal cancer and melanomas.
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Method name and description
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Polymerase chain reaction (PCR) followed by DNA sequencing is performed using primers specific for coverage of exon 2 (codons 1-34), exon 3 (codons 42-89) and exon 4 (codons 96-150) of the NRAS gene. The resulting PCR products are purified and sequenced to detect NRAS mutations. Sequence analysis is performed using Big Dye Terminator chemistry and detected using Genetic analyzer The resulting sequences were compared with the NCBI reference sequence (NM_002524).
In addition, Real Time – PCR based assay is used for detection of NRAS hot spots in exon 2, 3 and 4 of NRAS gene.
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Reporting name
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NRAS mutation analysis:
Detected indicates presence of NRAS mutation.
No mutation detected: indicates absence of NRAS mutation.
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Clinical information
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The analysis of NRAS status is considered medically necessary to predict treatment responses and prognosis in several cancer types. Activating mutations in NRAS lead to constitutive RAS signaling, the final consequence being cell growth and potential transformation through activation of the MEK/ERK pathway. Thus, NRAS mutations can be an early event in tumor initiation and are believed to be driver mutations associated with oncogenesis.
Cutaneous melanoma is known to harbour activating mutations of the NRAS gene in 15 to 20% of primary tumors and this mutation persists throughout metastasis spread. By far, the most common site of NRAS mutation is codon 61 in exon 3, with reported frequencies ranging from 4% to 50%. Of these, NM_002524: c.181C>A (Gln61Lys) and c. 182A>G (Gln61Arg) occur the most frequently. Mutations are also known to occur in codons 12 and 13 in exon 2, but at a much lower frequency.
NRAS mutations in exons 2, 3 and 4 occur in ~ 1-6% of colorectal cancers, and it has been observed that wild type NRAS together with wild type BRAF and KRAS is associated with response to EGFR antibody therapy. Literature has also demonstrated patients with NRAS-mutated colorectal tumors are less likely to respond.
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Specimen type / Specimen volume / Specimen container
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FFPE tissue:
Option 1: Tissue sections of 10X (5-7 µm) fixed on non-charged slides with an H&E stained slide with marked tumor area.
Option 2: Tissue curls of 6 sections (10 µm) collected in 2 mL tube (e.g. Eppendorf).
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Collection instructions / Special Precautions / Timing of collection
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Specimens are arranged from Sunday to Wednesday from the Anatomical Pathology Department to the Molecular Genetics Laboratory at room temperature.
Please avoid direct sun light exposure.
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Relevant clinical information to be provided
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The following points must be provided:
- The tumor rich area on the must be marked by a consultant pathologist.
- Tumor percentage.
- Cancer type.
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Storage and transport instructions
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The specimens are stored and transported at Room Temperature (16 - 25° C).
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Specimen Rejection Criteria
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Consistent with Diagnostic Genomic Division policies
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Biological reference intervals and clinical decision values
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For Real Time - PCR: Detected amplifications of the mutant target with mutant allele frequency >5% are referred as Detected.
No amplification of the mutant allele due to low mutant allele frequency (<5%) or due to absence of mutant target are referred as No mutation detected.
For DNA sequencing: the lower limit of detection is >20%.
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Factors affecting test performance and result interpretation
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This test was validated for DNA extraction from FFPE, the FFPE process has effect on the DNA quality and might result on DNA degradation. In addition, PCR inhibitors due to FFPE fixation might affect the PCR amplification.
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Turnaround time / Days and times test performed / Specimen retention time
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Days Test is Performed :
Arranged by laboratory Rota
TAT :
Single gene testing: 10 working days.
Reflex testing: 10 working days for the first test and an additional week (5 working days) for every following test.
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