KRAS TARGETED MUTATION ANALYSIS
Label Name: KRAS
Lab Discipline: Molecular Diagnostics
Institution:  Duke University Health System 
EAP ID:  LAB6612 
Last Review:  3/17/2017 10:17:10 AM
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
  Formalin fixed paraffin embedded tissue blocks and slides can be sent to the lab at ambient temperature.
Causes for Rejection
  All:
  • Testing will not be performed on samples which contain too little tumor or too many non-neoplastic cells to reliably detect KRAS mutations.

Turn Around Time -  Routine: 14 days   Stat: N/A
Reference Values
KRAS
NO MUTATION DETECTED
Methodology
  This assay uses PCR with Locked Nucleic Acid (LNA)clamping followed by Sanger DNA sequencing to detect activating mutations in codons 12 and 13 within exon 2 of the KRAS oncogene. An H&E stained slide for each case is first evaluated to identify the regions of greatest tumor content. These regions are then macro-dissected from adjacent unstained formalin-fixed paraffin-embedded sections and used to prepare genomic DNA. The protein coding and flanking intronic sequences of exon 2 of the KRAS gene is amplified from this purified genomic DNA. An LNA probe is included to suppress amplification of wild type sequences. The primers used in these PCR reactions contain M13 universal primer “tails” at their 5’ ends, and have 3’ ends that are complementary to their genomic target sequence. The resulting PCR products are treated with an exonuclease/ phosphatase mixture (ExoSAP-IT) to remove excess PCR primers and nucleotides. These purified DNA amplicons are then sequenced using universal M13 forward and reverse sequencing primers (M13 Forward/-20 and M13 Reverse/-27) and the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystem). The products of the completed sequencing reactions are purified with the Big Dye XTerminator Purification Kit and resolved using the ABI 3130xl Genetic Analyzer. Data is analyzed using the ABI Data Collection software v3.0, Sequencing Analysis software 5.2 and SeqScape software v2.6. Sequences are compared to the reference DNA sequence .(GenBank Accession: NM_004985.3) for the KRAS gene.

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
    BACKGROUND:

The v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) maps to chromosome 12p12.1 and encodes a cytoplamsic membrane bound GDP/GTP-binding protein from the RAS family. KRAS 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 KRAS results in the activation numerous signaling pathways that regulate cell proliferation, differentiation, and apoptosis. Under normal conditions, KRAS activation is short-lived and regulated by the hydrolysis of GTP. However, specific single amino acid substitutions in codons 12 and 13 of KRAS generate a constitutively active GTP-bound protein. Constitutive activation of KRAS transforms the gene into a highly effective oncogene that plays a role in many human cancers, including pancreatic adenocarcinoma, non-small cell lung carcinoma, and colorectal adenocarcinoma.

CLINICAL SIGNIFICANCE AND UTILITY:

Approximately 30-40% of colorectal cancers (CRC) and 40-50% of metastatic colorectal cancers (mCRC) contain an acquired activating mutation in codon 12 or 13 of the KRAS oncogene. In patients with codon 12 or 13 KRAS-mutated mCRC, EGFR-based antibody therapies ERBITUX (cetuximab) and Vectibix (panitumumab) have little to no efficacy. The Food and Drug Administration (FDA) notes that "retrospective analyses of metastatic colorectal cancer trials have not shown a treatment benefit for the EGFR inhibitors in patients whose tumors had KRAS mutations in codon 12 or 13 and that the use of the drugs is not recommended for the treatment of colorectal cancer patients with these mutations." In contrast, patients with tumors that lack KRAS mutations have a response rate to these drugs which ranges from 17% to 41%. Thus, KRAS mutation analysis can be used to determine which patients with mCRC would benefit most from cetuximab and panitumumab. A recent report suggests that tumors with the G13D mutation may be more sensitive to EGFR-based antibody therapies than tumors containing other mutations. This study, however, is limited by a relatively small sample size (Thirty two G13D KRAS mutant tumors). Additional studies are likely necessary to confirm this finding.

In addition to its utility in colorectal cancer, KRAS mutation analysis can be useful in decision making in non-small cell lung cancer patients. Activating K-RAS mutations are present in approximately 20% of patients with non small-cell lung carcinoma. Meta analyses of the published literature have shown a correlation between K-RAS mutation status and response to EGFR targeted therapies. However, data are complicated by the low clinical sensitivity of K-RAS mutations for determining responsiveness to therapy. Interpretation is further complicated by the interplay between K-RAS mutations and EGFR mutations, both of which have been implicated in lung carcinoma. Mutations in EGFR and KRAS tend to be mutually exclusive, but this is not always the case.

Multiple factors including clinical findings, tumor histology, ethnicity, gender, smoking status and the results of other laboratory tests also contribute to utility, efficacy and appropriateness of EGFR targeted therapies in treating lung and colon cancer patients. Thus, 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.

INTERPRETATION:

Sequence variants will be compared to those referenced by the available literature and recorded in the MDL database. Individual known polymorphisms may not be reported. Sequence changes will be reported as: (1) sequence variation is previously reported as an activating mutation, (2) sequence variation is previously unreported and is of unknown significance, (3) sequence variation is previously unreported and is likely not significant, or (4) sequence variant is previously reported as a benign polymorphism. At the discretion of the director, the significance of a sequence change may be speculated upon based on the type of variant, its position in the gene, and its effect on the amino acid sequence/protein. (5) In the case of an indeterminate sample result due to a variant detected in both forward and reverse reactions below the assay sensitivity of ~5%, at the discretion of the laboratory director the sample may be subject to additional testing to confirm or rule out a mutation.

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

Benesova L, et al., Multiplicity of EGFR and KRAS mutations in non-small cell lung cancer (NSCLC) patients treated with tyrosine kinase inhibitors, Anticancer Res. 2010; 30:1667-1671.

De Roock W et al. "Association of KRAS p.G13D mutation with outcome in patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab." JAMA. 2010; 304(16):1812-1820.

De Roock 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. 2007; 19(3):508-515.

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

Jackson MA, Lea I, Rashid A, Peddada SD, and Dunnick JK. Genetic Alterations in Cancer Knowledge System: Analysis of gene mutations in mouse and human liver and lung tumors. http://tools.niehs.nih.gov/gac/datamining/genetics. Toxicological Sciences. 2006; 90(2):400-418.

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

Linardou H, et al., Assessment of somatic k-RAS mutations as a mechanism associated with resistance to EGFR-targeted agents: a systematic review and meta-analysis of studies in advanced non-small-cell lung cancer and metastatic colorectal cancer, Lancet Oncol. 2008; 9:962-972.

Murray S, et al., Somatic mutations of the tyrosine kinase domain of epidermal growth factor receptor and tyrosine kinase inhibitor response to TKIs in non-small cell lung cancer: an analytical database, J Thorac Oncol. 2008; 3:832-839.
   
Indications
    KRAS mutation analysis can be used to predict the efficacy of EGFR based therapies in colorectal and non-small cell lung carcinoma patients.
   
Contraindications
    None
   
Limitations
    The sensitivity and specificity of DNA sequencing is high for the detection of nucleotide base changes, small deletions, and insertions in the regions analyzed. This assay may not detect an acquired mutation in codons 12 or 13 which is present below the 5% detection limit (i.e., a neoplastic cell population comprising less than 5% of total cells in the sample). Only amino acids 1-37 of the KRAS gene were examined. Changes outside of this region 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. For additional information or for help interpreting the results of this test, clinicians should contact the DUHS Clinical Molecular Diagnostics Laboratory. Patients should contact their healthcare provider with any questions related to this report.
   
Test Synonyms
  Synonym(s): Cetuximab
Synonym(s): Colorectal cancer
Synonym(s): CRC
Synonym(s): EGFR
Synonym(s): KRAS
Synonym(s): Non Small Cell Lung Cancer
Synonym(s): NSCLC
Synonym(s): Panitumumab
Molecular Diagnostics Laboratory
(MDX)

Medical Director:
 Michael Datto, M.D., Ph.D.
 Phone: 919-684-6965
 Email: michael.datto@duke.edu
Lab Director:
 Catherine Rehder Ph.D, FACMG
 Phone: 919-613-8434
 Email: catherine.rehder@duke.edu
Lab Director:
 Siby Sebastian Ph.D., DABMG
 Phone: 919-613-8432
 Email: siby.s@duke.edu

Address: 
 Wadsworth Bldg, Cytogenetics, Rm 0220
 2351 Erwin Rd
 Durham,  NC  27705
 Phone: 919-684-2698
 FAX: 919-668-5424

Performing Times: