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The development of drugs that target proteins that cause cells to proliferate has revolutionized the treatment of several cancers over the last decade. Meanwhile, the study of the pathways and mutations involved in tumor formation and progression has advanced our knowledge of potential drug targets, providing a better understanding of both the mechanisms of action and resistance of these drugs. This has resulted in the expanding and important role that molecular testing plays in identifying those patients most likely to benefit from these new drug treatments. A prime example is the role that KRAS mutation analysis plays in determining which patients with colorectal or lung cancer are eligible for anti-epidermal growth factor receptor (anti-EGFR) therapies.
Mechanism of Action
The epidermal growth factor receptor (EGFR)—also known as HER1 and ERBB1—belongs to the ErbB family of transmembrane tyrosine kinase receptors. EGFR is a dimeric transmembrane receptor that triggers several signaling cascades. (Figure 1A) The activating phosphorylation signal initiated by the intracellular active site of EGFR triggers the KRAS-BRAF-MEK-ERK pathway via growth factor receptor-bound protein 2 (GRB2) and son of sevenless (SOS), and activates KRAS by a GTPase-coupled reaction. The PI3K/AKT pathway also is triggered by EGFR activity. EGFR–signaling cascades play an important role in cell proliferation, angiogenesis, cell migration, cell survival, and cell adhesion. These cellular processes are often abnormal in malignant cells as a result of mutations in many of the genes in these pathways.
Two classes of drugs are now available that target EGFR. The first class includes panitumumab (Vectibix by Amgen), a fully humanized monoclonal antibody, and cetuximab (Erbitux by Bristol-Myers Squibb), a chimeric (mouse/human) antibody. The second class includes the small molecule tyrosine kinase inhibitors gefitinib (Iressa by AstraZeneca) and erlotinib (Tarceva by Genentech/OSI Pharmaceuticals and Roche). Both types of drugs inhibit downstream signaling triggered by EGFR:
Anti-EGFR drugs have shown promising results but cost approximately $100,000/patient/year depending on dose and duration of treatment.2 In addition, EGFR inhibitors can cause side effects including hypersensitivity reactions.3
While initial clinical trials using anti-EGFR drugs for non–small cell lung cancer were encouraging because some patients showed a significant clinical response, other studies did not show improved efficacy in combination with other chemotherapeutic agents.4 Interestingly, favorable responses to anti-EGFR therapies in lung cancer were primarily observed in patients with mutations of EGFR.6 Subsequent studies, however, demonstrated lung cancers with mutations in the KRAS gene (a member of the EGFR pathway) were associated with a group of patients whose tumors did not respond to anti-EGFR therapy. (Figure 1D) Because EGFR mutations do not appear to be as common in colorectal adenocarcinomas as in lung cancer, they are not used to predict response to anti-EGFR therapy for colorectal cancer. However, the same KRAS mutations that predict a lack of response to anti-EGFR therapy for lung cancer also predict a lack of response for colorectal cancer.5 Thus, testing for KRAS mutations provides a strategy for selecting lung and colorectal cancer patients most likely to respond to anti-EGFR drugs.5,6 Basing decisions on molecular testing enables physicians to provide individualized medicine in selecting the most effective chemotherapeutic regimens for each patient and limiting the associated risks and costs. As a result, both the National Comprehensive Cancer Network (NCCN) and the American Society of Clinical Oncology (ASCO) have recently issued new guidelines and updated recommendations for KRAS mutation analysis for patients being considered for anti-EGFR therapy.7,10
Several genes that encode proteins in the EGFR– signaling cascade are known to harbor mutations in tumors such as colorectal and lung cancer. In particular, activating mutations (ie, mutations that increase the activity of the oncogenic protein) of KRAS commonly occur in these tumors. These KRAS mutations are almost always found in codons 12 and 13 in exon 2, but occasionally involve codon 61 in exon 3 or other codons. The activated KRAS results in downstream signaling that is independent of its normal upstream EGFR regulation. KRAS activating mutations negate the mechanism of action of the anti-EGFR class of drugs by stimulating the EGFR–signaling cascade at a point downstream of the drug’s target. The association between KRAS mutations and a failure to respond to anti-EGFR therapies has been observed for both monoclonal antibodies and small molecule tyrosine kinase inhibitors.1,5,6
KRAS mutations are frequently found in a number of neoplasms. However, the relationship between KRAS mutations and drug response of anti-EGFR therapies has been most extensively studied in lung and colorectal cancer. Lung cancer is the second most common cancer in both sexes, with an estimated 215,020 new cases in 2008 resulting in 161,840 deaths. Colorectal cancer is the third most common cancer in both sexes, with an estimated 148,810 new cases in 2008 resulting in 49,960 deaths.11 KRAS mutations are identified in 15% to 30% of pulmonary adenocarcinomas and in 30% to 40% of colorectal (CRC) adenocarcinomas.1 Given the relatively high frequency of KRAS mutations in these tumors, mutation analysis can have a significant impact on the number of patients who are, or are not, likely to respond to anti-EGFR therapy due to the presence of a KRAS mutation.
For many years, classification of pulmonary tumors centered on determining whether the patient had a small cell or non–small cell tumor since therapy was largely based on this distinction. However, with the development of chemotherapeutic regimens that incorporate anti-EGFR drugs, this characterization is no longer sufficient. Distinctions within the non–small cell group (which includes adenocarcinoma, squamous cell carcinoma, and large cell carcinoma), in particular, are increasingly relevant since adenocarcinomas are more likely respond to anti-EGFR therapy.6 The histologic type of colorectal and lung cancer for which anti-EGFR therapy and KRAS mutation testing is relevant is adenocarcinoma. NCCN guidelines currently recommend anti-EGFR drugs as first-, second-, or third-line therapy in advanced metastatic colon and rectal adenocarcinoma and advanced or metastatic pulmonary non–small cell carcinoma, if KRAS wild-type.7,9,10
Mayo Clinic’s Molecular Genetics Laboratory has recently introduced a KRAS mutation analysis assay (#89378 KRAS Gene, 7 Mutation Panel, Tumor Tissue) that tests for the common mutations in codons 12 and 13. (Table 1) Formalin-fixed, paraffin-embedded (FFPE) tissue is the preferred specimen, since a review of a hematoxylin and eosin (H&E)-stained section of the tissue being analyzed is required prior to mutation testing. The histologic review is done to select areas of the FFPE sample for macrodissection that have a sufficient quantity and concentration of tumor cells for analysis.
|Codon||DNA change||Amino acid change|
Since therapy is currently recommended only for colorectal and non–small cell lung cancer patients with advanced disease, the tissue of choice for testing is generally the metastatic lesion. However, metastatic tissue is frequently not available or is present in insufficient quantity for testing and, as a consequence, the primary tumor from the patient may be submitted for analysis. KRAS mutations are generally considered to be early events in the development of colorectal and lung cancer and—if present at all—would be expected to be present in both the primary and metastatic tumor. Overall, studies have shown that the concordance between the presence or absence of KRAS mutations in a patient’s primary tumor and corresponding metastatic tumors is high. Still, a small number of cases have been reported in which the KRAS mutation status of the primary and metastatic tumor, or between metastases, do not match.12 Thus, KRAS testing of the primary tumor may, on occasion, produce a result that may not indicate the mutation status in the metastatic lesion and may, therefore, not accurately predict systemic response to anti-EGFR therapy.
Direct sequencing, such as Sanger sequencing and pyrosequencing, is generally considered the “gold standard” for KRAS mutation detection. Although this method can detect any alteration within the amplified region, it requires a higher percentage of tumor DNA to reliably detect the mutation.13 Sanger sequencing of exon 2 is one of 2 methods used in Mayo Clinic’s Molecular Genetics Laboratory to detect KRAS mutations. (Figure 2)
A number of allele-specific methods also are available for KRAS mutation analysis. These assays have specific primers that allow for the detection of each of the common KRAS mutations. Although allele-specific PCR can only detect the alterations for which the specific primers are designed, these assays are generally more sensitive than DNA sequencing, detecting as little as 1% to 10% of mutant DNA from all the DNA in the sample being tested. The Thera Screen KRAS kit (DxS Ltd, Manchester, United Kingdom), which is used in the Molecular Genetics Laboratory for KRAS mutation detection, uses this strategy.13 (Figure 3)
An important consideration is that while KRAS mutations predict that a colorectal or lung cancer will not respond to anti-EGFR therapies, only a subset of tumors that lack a KRAS mutation will respond to anti-EGFR therapy. Growing evidence indicates that genes in the EGFR signaling pathway other than KRAS may also have mutations that predict whether a tumor will or will not respond to EGFR-blocking agents. For example, a V600E mutation in the BRAF oncogene (occurs in 4% of CRC adenocarcinomas) has, like KRAS mutations, been shown to predict a failure to respond to anti-EGFR therapies.14 BRAF V600E tumor testing also is available from Mayo Medical Laboratories as #87980, BRAF Mutation Analysis (V600E). Conversely, studies have shown that favorable responses to gefitinib in non–small cell lung cancer occur primarily in patients with specific mutations of the EGFR gene.15 In time, a panel of such tests may be necessary to provide the most accurate prediction of tumor response to anti-EGFR drugs.
Research in oncologic pharmacogenomics is growing rapidly. As understanding of mutations that can alter anti-EGFR drug response evolves, the reasonable expectation is that better testing algorithms and treatment guidelines for lung and colorectal cancer, as well as for other tumors that also harbor mutations in members the EGFR signaling cascade, will be developed. This area of molecular diagnostics is also growing due to the development of drugs targeted at receptors and molecules other than EGFR. While this new knowledge will enhance the ability to effectively treat patients, it will also complicate the testing algorithms necessary to determine the appropriate patients for these therapies. The number of tests being performed, often on a minimal amount of tissue, will continue to challenge the clinical laboratory and highlight the necessity to develop even more sensitive tests. Despite these challenges, the use of KRAS testing, to help identify which patients will benefit most and avoid the side effects and costs of anti-EGFR therapies, is an important step in the realizing the central goal of individualized medicine: the right drug for the right patient at the right time.
Authored by Rumilla K, Medeiros F, Halling KC, Thibodeau, S