1. KRAS testing in colorectal cancer: an overview

2. 2 What is KRAS? KRAS is a gene that encodes one of the proteins in the epidermal growth factor receptor (EGFR) signaling pathway This signaling pathway is important in the development and progression of cancer

3. 3 KRAS and the EGFR pathway The EGFR signaling pathway is activated in response to ligand binding to the cell- surface receptors: these ligands include TGFα and EGF The signaling cascade that is activated is involved in regulating genes that control cell cycle progression Cell cycle progression gives rise to tumor survival, growth and proliferation as well as metastasis and angiogenesis In the early part of the signaling cascade, the protein KRAS regulates downstream proteins involved in these effects The KRAS protein plays a central role in tumor development, regulating downstream proteins that are involved in proliferation, survival, metastasis and angiogenesis

4. 4 The EGFR pathway and the importance of KRAS status The KRAS gene may be normal (wild- type) or mutated Wild-type KRAS protein is active for a short period when the EGFR is stimulated The effects of the protein are closely controlled When KRAS is mutated the protein is permanently turned on, even without being triggered by EGFR-mediated signaling The effects of KRAS that lead to tumor growth and spread continue unregulated The KRAS status of a tumor may be indicative of prognosis and predictive of response to certain drugs

5. 5 Use of anti-EGFR antibodies to disrupt the signaling pathway Monoclonal antibodies against EGFR block signaling by the receptor and inhibit downstream events, including effects mediated by KRAS When KRAS is mutated and permanently switched on, blocking EGFR will probably not affect downstream events The tumor may continue to grow, proliferate and spread Therefore, blocking EGFR with a monoclonal antibody will be more effective in KRAS wild-type tumors In metastatic CRC, up to 65% of patients have KRAS wild-type tumors There are six KRAS mutations relevant to CRC at codons 12/13: G12A, G12V, G12D, G12C, G12S, G13D and G12R

6. 6 Biomarkers and KRAS testing There has recently been increased interest in the role of biomarkers in oncology, including the role of KRAS status as a biomarker in CRC and other EGFR-associated cancers The test for KRAS status uses standard methodology and is performed on a sample of tumor tissue (fresh frozen or paraffin- embedded) sent to a laboratory for analysis Determining the KRAS status of a tumor helps the treating physician to choose the most effective therapeutic approach for each individual patient Patients diagnosed with metastatic CRC should be tested for KRAS status to allow the optimum treatment strategy to be implemented

7. 7 The KRAS test A tumor sample is taken and sent to the laboratory – the test can use fresh, frozen or paraffin-embedded tissue A pathologist confirms that the tissue is cancerous, and a sample of DNA is prepared for the KRAS test The polymerase chain reaction (PCR) is used to amplify the DNA and test for KRAS mutation status* *Direct DNA sequencing can also be used

8. 8 There are commercially available PCR assays for KRAS KRAS tests are based on well-established quantitative PCR (qPCR) technology The probes used in this technique are specific for KRAS mutations known to confer constitutive activation The assays are highly specific The assays are robust and reliable Direct DNA sequencing can be used as an alternative to PCR

9. 9 Benefits of KRAS testing Knowing the KRAS status of the patient’s tumor allows treatment to be tailored to the individual Patients with metastatic CRC with KRAS wild-type tumors gain more benefit when treated with agents targeted to the EGFR than patients with KRAS mutant tumors 1,2 Anti-EGFR therapies may have activity independent of KRAS status An lgG1 anti-EGFR monoclonal antibody has been shown to stimulate antibody-dependent cellular cytotoxicity 3 Consequently this agent may offer some benefit in patients with KRAS mutant tumors Testing for KRAS status allows a tailored treatment strategy to be implemented 1) Bokemeyer et al. ASCO Annual Meeting 2008: abstract 4000. 2) Van Cutsem et al. ASCO Annual Meeting 2008: abstract 2. 3) Kimura et al. Cancer Sci 2007;98:1275-80