Colorectal cancer KRAS — Labs

 

KRAS therascreen - Kit US Version
The therascreen KRAS RGQ PCR Kit reliably detects mutations in the KRAS gene, which are found frequently in colorectal cancer. According to the US product information for ERBITUX, KRAS status evaluation using an FDA-approved test is used to determine treatment, and the therascreen KRAS test is the only test with this status.
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The therascreen KRAS test:

  • Is the only FDA-approved test for KRAS mutation detection
  • Is approved based on Phase III clinical trial data with ERBITUX of patients with KRAS wild-type tumors.
  • Is a standardized assay for reproducible results
  • Belongs to an easy and complete workflow with automated reporting

QIAGEN has a broad portfolio of companion diagnostic products in development.

Assay principle
Applications
Performance characteristics
therascreen assays in development
Reimbursement
Find out about ERBITUX for your patients
Back to topAssay principle

The therascreen KRAS test provides 8 separate PCR amplification reactions: 7 mutation-specific reaction in codons 12 and 13 of exon 2 of the KRAS oncogene, and a wild-type control in exon 4. The principle components of the kit are explained below.

Mutation reaction mixes

Each mutation-specific reaction mix uses a mutation-specific ARMS primer to selectively amplify and enrich the mutated DNA, and then uses a Scorpions primer to detect the mutation during a second amplification reaction (see ARMS Scorpions technology).

ARMS

Allele-specific amplification is achieved by ARMS, which exploits the ability of Taq DNA polymerase to distinguish between a match and a mismatch at the 3' end of a PCR primer. When the primer is fully matched, the amplification proceeds with full efficiency. When the 3' base is mismatched, only low-level background amplification occurs. Therefore, a mutated sequence is selectively amplified even in samples where the majority of the DNA does not carry the mutation.

Scorpions

Detection of amplification is performed using Scorpions. Scorpions are bi-functional molecules containing a PCR primer covalently linked to a probe. The probes incorporate the fluorophore carboxyfluorescein (FAM) and a quencher. The latter quenches the fluorescence of the fluorophore. When the probe binds to the ARMS amplicon during PCR, the fluorophore and quencher become separated, leading to a detectable increase in fluorescence.


Back to top Applications

Mutations in the KRAS oncogene are frequently found in human cancers (2–4). The presence of these mutations correlates with a lack of response to certain EGFR inhibitor cancer therapies in patients with metastatic colorectal cancer (6–7, 11–13). Such mutations in the KRAS oncogene are present in around 40% of cases. (5). Using Scorpions (8) and ARMS (Allele Refractory Mutation System) technologies (9–10), the therascreen KRAS test enables detection of the seven mutations in codons 12 and 13 of the KRAS oncogene against a background of wild-type genomic DNA (see Table 1). Based on data (1) in the COSMIC database (2012 v59), the seven mutations detected by the test account for >97% of all reported KRAS mutations in CRC patients.

Table 1. List of mutations and COSMIC identities*
 Mutation Base change COSMIC ID
 GLY12ALA (G12A) GGT>GCT 522
 GLY12ASP (G12D)  GGT>GAT 521
 GLY12ARG (G12R) GGT>CGT  518
 GLY12CYS (G12C) GGT>TGT 516
 GLY12SER (G12S) GGT>AGT 517
 GLY12VAL (G12V) GGT>GTT 520
 GLY13ASP (G13D) GGC>GAC 532
* COSMIC IDs are taken from the Catalog of Somatic Mutation in Cancer (www.sanger.ac.uk/genetics/CGP/cosmic).

References
  1. Catalog of Somatic Mutations in Cancer. Available at: www.sanger.ac.uk/genetics/CGP/cosmic.
  2. Hilger, R.A., Scheulen, M.E., Strumberg, D. (2002) The Ras-Raf MEK-ERK  pathway in the treatment of cancer. Onkologie 25, 511.
  3. Bachireddy, P., Bendapudi, P.K., Felsher, D.W. (2005) Getting at MYC through RAS. Clin. Cancer Res. 11(12), 4278.
  4. Benvenuti, S., et al. (2007) Oncogenic activation of the RAS/RAF signaling pathway impairs the response of metastatic colorectal cancers to anti-epidermal growth factor receptor antibody therapies. Cancer Res. 67 (6), 2643.
  5. De Roock, W., et al. (2007) KRAS mutations preclude tumor shrinkage of colorectal cancers treated with cetuximab. J. Clin. Oncol. 25 (18S), 4132.
  6. Neumann, J., Zeindl-Eberhart, E., Kirchner, T., Jung, A. (2009) Frequency and type of KRAS mutation in routine diagnostic analysis of metastatic colorectal cancer. Pathol. Res. Pract. 205 (12), 858.
  7. Di Fiore, F., et al. (2007) Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by Cetuximab plus chemotherapy. Br. J. Cancer 96, 1166.
  8. Newton, C.R. et al. (1989) Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res. 17 (7), 2503.
  9. Whitcombe, D., Theaker, J., Guy, S.P., Brown, T., Little, S. (1999) Detection of PCR products using self-probing amplicons and fluorescence. Nat. Biotechnol 17, 804.
  10. Thelwell, N., Millington, S., Solinas, A., Booth, J., Brown, T. (2000) Mode of action and application of Scorpion primers to mutation detection. Nucleic Acids Res. 28 (19), 3752.
  11. Bokemeyer, C., et al. (2008) K-Ras strategy and efficacy of first-line treatment of patients with metastatic colorectal cancer (mCRC) treated with FOLFIRI with or without cetuximab: The OPUS experience. J. Clin. Oncol. 26 (suppl), abstract 4000.
  12. Van Cutsem, E., et al. (2008) K-RAS status and efficacy in the first-line treatment of patients with metastatic colorectal cancer (MCRC) treated with FOLFIRI with or without cetuximab: The CRYSTAL experience. 26 (suppl), abstract 2.
  13. Tejpar, S. et al. (2008) Relationship of efficacy with K-RAS status (wild type versus mutant) in patients with irinotecan-refractory metastatic colorectal cancer (mCRC), treated with irinotecan (q2w) and escalating doses of cetuximab (q1w): The EVEREST experience (preliminary data). J. Clin. Oncol. 26 (suppl), abstract 4001.

Back to top Performance characteristics

The therascreen KRAS test belongs to an easy and complete workflow with automated reporting.

The therascreen KRAS test provides:

  • Turn-around-time — testing can be performed in < 8 hours
  • Standardized workflow and software for automated report generation

Performance characteristics
Item Characteristic
Tissue section 5 µm section
Tumor content required >20% tumor area (macrodissect 1 or more slides if ≤20%)
Mutation coverage 7 main mutations in codons 12 and 13
Time to result Testing can be performed in < 8 hours
Control/result interpretation Automatic
Limit of Detection — FFPE samples 0.8–6.4% mutant DNA


Back to top therascreen assays in development

The Rotor-Gene Q MDx offers one platform for all future QIAGEN companion diagnostic (CDx) assays:

  • Rotor-Gene Q MDx instrument was FDA-approved as part of the KRAS testing system
  • Rotor-Gene Q MDx is a key element in the QIAGEN products

Building the leading Companion Diagnostics platform, future IVD kits for submission to FDA include:

  • EGFR
  • JAK-2
  • PI3K
  • Others

therascreen KRAS RGQ PCR Kit — for in vitro diagnostic use 


Back to top Reimbursement

The therascreen KRAS test is:

  • The only FDA-approved test for KRAS mutation detection
  • Approved based on Phase III clinical trial data with ERBITUX of patients with KRAS wild-type tumor
  • A standardized assay for reproducible results
therascreen KRAS testing enjoys widespread coverage

Public and private payers endorse the value of KRAS testing with strong coverage policies for approved indications. Below is a sample of payers that have published positive coverage policies for KRAS testing.

Payers that have published positive coverage policies for KRAS testing
Sample listing
Aetna Blue Shield of CA
Anthem/Wellpoint Healthnet
BCBS AL Highmark BCBS
BCBS FL Humana
BCBS IL United Healthcare
BCBS MI Palmetto GBA (Medicare)
BCBS TX  

Public and private payers recognize the value of KRAS testing in determining the appropriateness of ERBITUX (cetuximab) for a specific patient. The following payers require that a KRAS test be performed prior to prescribing.

Payers that require a KRAS test prior to prescribing
Private payers Medicare payers
Aetna CGS
Anthem/Wellpoint CGS Medicare
BCBS AL FCSO Medicare
BCBS FL Palmetto
Cigna WPS Medicare
Empire BCBS  
Horizon BCBS  
Humana  

To confirm individual patient benefits please call QIAGEN Reimbursement Solutions at: 877-7-MYQIAGEN (877-769-7424).

Representatives are available from 8:00 a.m. through 8:00 p.m. Eastern Time, Monday through Friday.

Important: Coverage information provided by QIAGEN is gathered from third-party sources and is presented for informational purposes only. Coverage for KRAS testing varies by patient and is subject to contracting, deductibles, and payment caps. This information does not guarantee payment and QIAGEN makes no warranty regarding this information, its completeness, accuracy, or timeliness. Payer policies are complex and change frequently, and providers are responsible for all decisions relating to coding and reimbursement submissions. QIAGEN recommends that you consult your payers, reimbursement specialists, and/or legal counsel regarding coding, coverage and reimbursement issues.


QIAGEN has a broad portfolio of companion diagnostic products in development.


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External Websites
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Find out more about ERBITUX (cetuximab).
Find out more about Vectibix (panitumumab)
Clinical practice guidelines in oncology for patients with cancer.