In January 2009, The American Society of Clinical Oncology (ASCO) issued their first provisional clinical opinion (PCO) recommending that all patients with mCRC who are candidates for anti-EGFR monoclonal antibody therapy should undergo testing for KRAS mutations and that patients with KRAS mutations in codons 12 or 13 should not receive anti-EGFR monoclonal antibody therapy as part of their treatment.85 The PCO is based on studies identified in searches of the MEDLINE database (through October 2008) and the 2008 ASCO Annual Meeting presentations by the Blue Cross and Blue Shield Association Technology Evaluation Center. Importantly, the PCO is therefore based on studies that used assays capable of detecting KRAS mutations in codons 12 or 13 only and did not include a comparison of the specificity or sensitivity of assays currently available for KRAS mutation analysis. In addition to the ASCO PCO, the National Comprehensive Cancer Network has updated its guidelines with a recommendation that therapies including panitumumab or cetuximab be limited to patients with wild-type KRAS for patients with advanced or metastatic colon or rectal cancer.86,87 The US Food and Drug Administration has recently approved revisions to the prescribing information for panitumumab and cetuximab that now state that their use is not recommended in patients with colorectal tumors that have KRAS mutations in codons 12 or 13.31,34
An important aspect in the determination of concordance between different assay techniques is the selection of a gold-standard method for the determination of reference standards. Such a method should be able to accurately quantify the amount of the specific analyte in question. Emulsion PCR used in combination with picotiter-plate sequencing offers rapid and sensitive screening of entire genomes or genome subregions and may represent such a gold standard in gene mutation testing.91 In this approach, developed by Roche Diagnostics Corporation/454 Life Sciences (Branford, CT, USA), specific genomic regions of interest are amplified by PCR with primers incorporating universal tags using PCR on DNA extracted from tissue [eg, formalin-fixed paraffin-embedded (FFPE) tissue], with specific linker sequences being added by a subsequent round of PCR, and then hybridized to beads using limiting dilutions to enable 1 amplimer to bind per bead. After clonal amplification by emulsion PCR, the beads are dispersed into a picotiter-plate and subjected to pyrosequencing, during which nucleotide incorporation is measured as luminescence. The technology enables multiple reads of each amplimer to be measured, thereby enabling accurate quantitation of the number of genome equivalents of wild-type or mutant amplimers. Thus, this technology can be used to accurately determine the number of mutant genome equivalents in a given sample, providing an accurate “gold standard” sample that can be used in the evaluation of other assays. A recent study assessing comparability between 4 commercial KRAS tests and an internal direct sequencing core laboratory using colorectal tumor samples found that some tests are in close agreement, but others are not.92 Similar results were observed in another study that compared KRAS testing techniques in clinical colorectal cancer samples.93 Furthermore, a recent study showed that cycle sequencing and real-time PCR were reliable and comparable methods for KRAS mutation testing, with the caveat that the sample must contain a sufficient proportion of tumor cells.94
Agreement among pathologists and oncologists on a standardized method of comparing and validating KRAS mutation testing methods is essential for the future implementation of KRAS testing in the clinic. Studies have showed some lack of comparability between assays.92 This lack of agreement is unsurprising, but it raises the question of how pathologists should assess concordance between KRAS tests or conduct proficiency testing. It has been proposed that concordance testing should consist of a 2-step process using 2 separate sets of samples. The first step would determine the ability of a laboratory to extract DNA from a range of FFPE tumor blocks in a form suitable for the assay used by the laboratory (typically PCR-amplifiable DNA). The second step would test the ability of the laboratory to identify mutations in sample extract mixtures that have accurately determined amounts of mutant and wild-type KRAS alleles. For example, one method for accurately determining the amount of mutant KRAS in an FFPE sample is emulsion PCR picotiter-plate sequencing as carried out on the Roche 454 sequencer91 that would enable an accurate ratio of mutant to wild-type alleles to be determined within a tumor extract. In addition, such extracts could be mixed with FFPE extracts containing only wild-type alleles to allow a range of samples to be generated, each with varying levels of mutant to wild-type alleles spanning a variety of mutant alleles and at levels close to the target sensitivity (cutoff) required for clinical utility. If prepared by a reference laboratory, these extracts could have widespread use.
Another important consideration for pathologists and the oncology community is the impact of mutations in other genes on clinical decision making. Even among patients with wild-type KRAS, a large proportion do not respond to EGFR-targeted monoclonal antibody therapies.4,98 Therefore, should routine pretreatment practice also include testing for mutations in other genes downstream of the EGFR, such as BRAF? In a retrospective analysis of patients with mCRC treated with panitumumab or cetuximab, BRAF V600E mutations resulted in a lack of response to treatment and significantly shorter progression-free survival and overall survival compared with wild-type BRAF.99 Another gene that may affect patient response to anti-EGFR therapy is PIK3CA. A recent study of patients with curatively resected colorectal cancer showed that PIK3CA mutations were associated with increased cancer-specific mortality [hazard ratio (HR), 2.23; 95% CI, 1.21-4.11]. The association between PIK3CA mutations and increased mortality was strongest among patients with wild-type KRAS (HR, 3.80; 95% CI, 1.56-9.27) but was not observed in patients with KRAS mutations (HR, 1.25; 95% CI, 0.52-2.96). Mutations in PIK3CA have also been shown to confer resistance to anti-EGFR therapies among patients with mCRC.100 In that study, among 110 patients with mCRC who received treatment with either panitumumab or cetuximab, 15 (13.6%) had mutations in PIK3CA. None of the patients with PIK3CA mutations had objective responses (P=0.038). In addition, PIK3CA mutations were associated with shorter progression-free survival (P=0.0035) compared with wild-type PIK3CA. Although further studies will be necessary to assess their utility as predictors of response to anti-EGFR therapy, it is possible that mutational analyses will extend beyond KRAS to include BRAF and PIK3CA.
The authors thank Ali Hassan, PhD, and Benjamin Scott, PhD (Complete Healthcare Communications, Inc., Chadds Ford, PA), whose work was funded by Amgen Inc. (Thousand Oaks, CA), for assistance in the preparation of this manuscript.
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