Advances in Anatomic Pathology:
KRAS Mutation Testing in Human Cancers: The Pathologist's Role in the Era of Personalized Medicine
Wang, Hanlin L. MD, PhD*; Lopategui, Jean MD*; Amin, Mahul B. MD*; Patterson, Scott D. PhD†
*Department of Pathology & Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles
†Amgen Inc., Thousand Oaks, CA
Hanlin L. Wang, Jean Lopategui, and Mahul B. Amin have no disclosures. S.D.P. is an employee and stockholder in Amgen Inc.
Reprints: Scott D. Patterson, PhD, Amgen Inc. One Amgen Center Drive, MS 38-3-A, Thousand Oaks, CA 91320 (e-mail: firstname.lastname@example.org).
A number of studies have shown that although antiepidermal growth factor receptor (EGFR) monoclonal antibodies are effective treatments for metastatic colorectal cancer (mCRC), only patients with wild-type KRAS tumors derive clinical benefit from these therapies. The anti-EGFR monoclonal antibodies panitumumab and cetuximab are approved in the United States for treatment of mCRC refractory to chemotherapy but are not recommended for use in patients with mutations in KRAS codons 12 or 13. Similarly, panitumumab is approved for the treatment of mCRC only in patients with wild-type KRAS in Europe and Canada. It is clear that KRAS mutational analysis will become an important aspect of disease management in patients with mCRC. Consequently, it will be important for pathologists and oncologists to develop and agree on standardized KRAS testing and reporting procedures to ensure optimum patient care. Pathologists will be central to this process because of their crucial role in selecting appropriate tumor specimens for testing, choosing the molecular diagnostic laboratory to be used, assisting in the selection of a suitable KRAS test, and interpreting the results of KRAS mutational analysis. Guidelines for KRAS testing that address these and other important points of consideration have recently been proposed in the United States and the European Union.
Improved understanding of the genetic events that underlie tumor development, progression, and metastasis has had a significant impact on research efforts directed toward treatments for patients with cancer.1 In particular, there has been a focus on the development of targeted therapies and on the use of molecular analyses to identify patients most likely to respond to these therapies. For example, the antiepidermal growth factor receptor (EGFR) monoclonal antibodies panitumumab and cetuximab have been shown to be effective therapies for metastatic colorectal cancer (mCRC), but, as with most cancer therapies, not all patients derive clinical benefit from these treatments.2,3 Genetic analyses have revealed that the presence of mutations in the gene KRAS (Kirsten ras) can predict lack of response to panitumumab and cetuximab in patients with mCRC.4–19 These observations will have significant implications for pathology practice. This article describes the role that mutations in KRAS play in the development of cancer, summarizes the impact of KRAS status on response to anti-EGFR therapies, and discusses currently proposed guidelines for KRAS mutation testing and reporting and the potential impact of these guidelines on pathology practice.
THE EPIDERMAL GROWTH FACTOR RECEPTOR AS A THERAPEUTIC TARGET
The EGFR is a clinically validated therapeutic target for several cancer types, including colorectal cancer, non–small-cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck, and pancreatic cancer.20 Epidermal growth factor (EGF) is one of several known EGFR ligands that also include transforming growth factor α, amphiregulin, epiregulin, and heparin-binding EGF.21 Binding of EGF to the EGFR results in receptor dimerization or, alternatively, heterodimerization with other members of the ErbB family.21,22 Subsequent recruitment of adaptor proteins to these dimer complexes results in the activation of various downstream signaling pathways, such as the Ras/Raf/mitogen-activated protein kinase pathway, the phosphatidylinositol 3-kinase/AKT pathway, and the signal transducer and activator of transcription pathway that are important inducers of transcriptional regulation of cell division, differentiation, migration, adhesion, and apoptosis.23–26 Dysregulation of the EGFR signaling pathway, which can be caused by constitutive activation of components of the signaling pathway and/or overproduction of receptors/ligands, can promote tumor growth, survival, and metastatic spread.21 Lung and colorectal tumors commonly express EGFR, often at higher levels than normal tissues.27–30
Panitumumab, a fully human anti-EGFR monoclonal antibody, is approved in the United States as a single agent for the treatment of mCRC refractory to fluoropyrimidine, irinotecan, and oxaliplatin chemotherapy regimens, but is not recommended for use in patients with colorectal tumors that have KRAS mutations in codons 12 or 13.31 Panitumumab is approved in Europe and Canada for the treatment of mCRC in patients with wild-type KRAS.32,33 Cetuximab, a chimeric anti-EGFR monoclonal antibody, is approved in the United States as a single agent in patients refractory to irinotecan-based chemotherapy and oxaliplatin-based chemotherapy or intolerant of irinotecan-based chemotherapy and in combination with irinotecan for the treatment of EGFR-expressing mCRC refractory to irinotecan alone.34 Like panitumumab, it is not recommended for use in patients with KRAS mutations in codons 12 or 13.34 Cetuximab has also been approved in the United States for the treatment of locally advanced squamous cell carcinoma of the head and neck (SCCHN) in combination with radiation therapy and for the treatment of recurrent or metastatic squamous SCCHN for patients who have disease progression after platinum-based therapy. Cetuximab is approved in Europe as a single agent or in combination with chemotherapy for the treatment of mCRC refractory to chemotherapy in patients with wild-type KRAS, in combination with radiation therapy for locally advanced SCCHN, and in combination with platinum-based chemotherapy for recurrent and/or metastatic SCCHN.35 Anti-EGFR monoclonal antibodies are also being investigated for other indications, including small-cell carcinoma of the head and neck, NSCLC, esophageal cancer, renal cancer, breast cancer, and pancreatic cancer.36 In addition to monoclonal antibodies, small-molecule EGFR tyrosine kinase inhibitors have been approved (eg, erlotinib for NSCLC) or are under investigation for treatment of a variety of solid tumor types.36,37
KRAS AND ITS SIGNALING PATHWAYS
The gene KRAS encodes KRAS, a member of the Ras family of small guanine nucleotide–binding proteins38–40 first identified as a cellular homolog of a transforming gene in the Kirsten rat sarcoma virus.39,41 KRAS is a key component of intracellular signaling pathways regulated by a broad variety of cell surface receptors, including the EGFR.42 KRAS activity (and consequently downstream signaling) is regulated by guanine exchange factors (GEFs) and guanosine triphosphatase–activating proteins (GAPs). KRAS is activated by exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) (Fig. 1); this process is promoted by GEFs.42,43 Like all Ras family members, the intrinsic GTPase activity of KRAS hydrolyzes GTP to GDP, thereby converting the enzyme back to its inactive GDP-bound form.40,44 This intrinsic GTPase activity of Ras proteins is markedly enhanced by GAPs.42,45
KRAS plays a key role in the EGFR signaling network.21,46 Activation of the EGFR promotes the activity of the GEFs,43 and in turn, GEFs activate KRAS.47 Ultimately, activation of the EGFR signaling pathway and KRAS results in alterations in expression of genes that regulate apoptosis, migration, growth, adhesion, and differentiation.21 Signaling pathways downstream of KRAS that are important for regulating tumor growth and progression include the RAF-MEK-ERK,48,49 mitogen-activated protein kinase,50 and PI3-K pathways.49,51
KRAS MUTATIONS IN HUMAN CANCERS
Activating mutations in KRAS have been observed at relatively high frequency in a variety of tumor types, including pancreatic cancer, NSCLC, and colorectal cancer.44 The frequencies of KRAS mutations in several types of human cancers recorded in the Catalogue of Somatic Mutations in Cancer database (COSMIC; Wellcome Trust Sanger Institute, Cambridge, UK)52 are shown in Table 1. The overwhelming majority of mutations in KRAS result in amino acid substitutions at codons 12 and 13.52,53 Activating mutations in members of the Ras family either inhibit the GTPase activity of the enzyme or render it insensitive to activation by GAPs, resulting in accumulation of the active GTP-bound conformation of the enzyme (Fig. 1).53,54 Essentially, these mutations render KRAS constitutively active, resulting in unregulated downstream signaling.53
KRAS mutations have been associated with poor prognosis in a number of tumor types, including pancreatic cancer, thyroid cancer, and NSCLC.55–57 In colorectal cancer, KRAS mutations are observed in approximately 35% to 40% of tumors,58–60 occurring most frequently in codons 12 and 13.60,61 Overall, mutations in codons 12 and 13 account for 98.5% of mutations in KRAS in colorectal cancer.60 Mutations in these codons have been associated in some trials with poor clinical outcome in patients with colorectal cancer,58,62–64 and have a particularly strong negative association with progression-free survival and overall survival among patients with Dukes stage C or D tumors.58,59 In addition, the presence of a glycine-to-valine mutation in codon 12 of KRAS exon 2 is associated with reduced progression-free survival and overall survival.58 To our knowledge, this is the only study showing the impact of a specific amino acid substitution in KRAS on patient prognosis. However, it is important to note that, in other studies, associations between KRAS status and clinical outcomes have not been observed.65 Thus, the utility of KRAS as a prognostic marker in colorectal cancer needs further investigation.
Several studies have shown that a large proportion of KRAS mutations appear relatively early (ie, adenoma stage) in colorectal tumorigenesis,61,66–69 although their presence is more frequently observed in adenomas of later stage and larger size.61,66 A very high proportion of nondysplastic aberrant crypt foci have also been observed to harbor KRAS mutations.66,69 Several studies have also shown that KRAS mutations present in primary colorectal tumors are also typically present in metastases (Table 2),70–78 indicating stability of KRAS mutations in the metastatic process. In some instances, the lack of agreement between these studies may have resulted from a lack of assay sensitivity or specificity rather than a true lack of concordance. Mutations in codons 12 and 13 of KRAS in primary colorectal tumors may be conserved in distant and local metastases for up to 5 years.72 In addition, the intratumoral genetic heterogeneity of point mutations and allelic losses observed in colorectal tumors seems to be more frequent during early tumor stages (90%) compared with later stages (67%)80 and provides another source of potential discordance when primary and metastatic samples are analyzed. Using improved tissue microdissection and DNA extraction techniques, Baisse et al showed that 67% of colorectal tumors were heterogeneous for more than equal to 1 genetic alteration.81 Furthermore, intratumoral genetic heterogeneity was more frequently observed for chromosomal loss of heterozygosity (58% to 67%) compared with point mutations in genes such as KRAS (20%).
RATIONALE FOR KRAS MUTATION TESTING IN EPIDERMAL GROWTH FACTOR RECEPTOR–TARGETED CANCER THERAPY
As KRAS plays an important role in EGFR signaling,21,46 recent studies have investigated the use of KRAS mutational status as a predictor of response to EGFR inhibitors. The most striking results have come from studies of patients with mCRC treated with either panitumumab or cetuximab. In a limited post hoc analysis of results from 533 patients enrolled in 3 phase 2 clinical trials in which patients refractory to prior chemotherapy received panitumumab, 62 patients who had tumor samples available were included in the analysis. Of these, 24 (39%) had mutations in KRAS at codons 12 or 13 but no patients had mutations at codon 61.12 Patients with wild-type KRAS had a better response to treatment with panitumumab than patients with mutations in KRAS. Best objective response (complete response or partial response per Response Evaluation Criteria in Solid Tumors or World Health Organization criteria) rate (11% vs. 0%), progression-free survival (16.2 vs. 7.4 wk), and overall survival (42.9 vs. 22.2 wk) all favored patients with wild-type KRAS compared with patients with mutations in KRAS. This study provided a sufficient level of confidence to pursue an analysis of samples from the pivotal phase 3 study in which a prespecified statistical analysis plan was used to determine whether the effect of panitumumab on progression-free survival differed by KRAS status, using an assay using allele-specific polymerase chain reaction (PCR) (DxS KRAS Mutation Test Kit, DxS Ltd, Manchester, UK). The DxS KRAS Mutation Test Kit is a well-defined assay that interrogates the 7 most common mutations in codons 12 and 13. The assay was validated according to Clinical and Laboratory Standards Institute guidelines that include evaluation of the performance characteristics of sensitivity, specificity, and precision, by a Belgian government–accredited laboratory. The pivotal phase 3 study compared panitumumab plus best supportive care in patients with mCRC refractory to prior treatment with chemotherapy. Of the 463 patients enrolled, 427 (92%) had tumor samples available for KRAS analysis with the DxS KRAS assay; testing was carried out in the central laboratory blinded to treatment assignment and clinical outcome. Overall, 43% of tumor samples had KRAS mutations at codons 12 and/or 13.4 The clinical benefits of panitumumab treatment were limited to patients with wild-type KRAS (Fig. 2).4 Among patients with wild-type KRAS, progression-free survival after treatment with panitumumab plus best supportive care or best supportive care alone was 12.3 weeks and 7.3 weeks, respectively. In contrast, among patients with mutations in KRAS, progression-free survival was 7.4 weeks and 7.3 weeks in the panitumumab plus best supportive care and best supportive care alone groups, respectively. Partial responses were observed in 17% of patients with wild-type KRAS who received panitumumab, but none were observed in patients with mutant KRAS who received panitumumab. An extensive analysis of the 3 phase 2 studies and the panitumumab-treated arm of the phase 3 study described above was then undertaken: assaying all available samples generated results across the studies for more than 90% of patients (ie, ascertainment of >90%). The results revealed that no objective responses were observed in 320 patients who had KRAS mutant tumors.82 Consistent results have been observed in a number of studies of associations between KRAS status and response to cetuximab or panitumumab (Table 3).4–19KRAS mutations have also been associated with poor response to erlotinib treatment in patients with NSCLC.57,83
The results described above show that mutations in KRAS are a powerful negative predictor of response to anti-EGFR therapy in patients with mCRC and illustrate the need for sensitive and accurate KRAS testing for these patients. Screening patients for KRAS mutations before the initiation of therapy may prevent unnecessary toxicity and healthcare expense in patients unlikely to respond to EGFR-targeted therapy. As with other monoclonal antibodies, costs associated with cetuximab and panitumumab treatment are substantial.84 Identifying patients with wild-type KRAS through mutational analysis may result in improvements in cost-effectiveness.84
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
ANALYTICAL SENSITIVITY AND SPECIFICITY OF COMMON KRAS TESTING PLATFORMS
Although it is now accepted that determination of KRAS status should play an important role in determining the utility of anti-EGFR therapy in patients with mCRC, it is less clear how KRAS status should be determined. A number of tests for KRAS mutations are available, the majority of which are laboratory based.88 Direct sequencing analysis and real-time PCR are commonly used tests for detecting KRAS mutations.89 Direct sequencing analysis is capable of detecting all possible mutations in exons 2 and 3 of KRAS but may lack sensitivity compared with other methods. Real-time PCR uses oligonucleotide primers that bind specifically to the most common mutations in codons 12 and 13 and, although highly sensitive as a result of preferential amplification of the mutant allele in large excess of the wild-type allele, will identify only those mutations targeted. Although it is possible to detect uncommon mutations (eg, codon 61 in mCRC12,55), tests that screen for rare mutations are generally unavailable. The TheraScreen KRAS mutation assay (DxS) is an allele-specific PCR assay that detects 7 common mutations in codons 12 and 13 of exon 2 for patients with mCRC who are candidates for panitumumab or cetuximab therapy. TheraScreen carries a valid CE mark and is approved for clinical use in the European Union, Canada, and Australia. A second assay carrying a valid CE mark and approved for clinical use in the European Union is the PyroMark kit (Qiagen Inc., Valencia, CA, USA). This pyrosequencing-based assay detects KRAS mutations in codons 12, 13, and 61 and is a more sensitive sequencing method than traditional Sanger sequencing. In the laboratory setting, pyrosequencing has been used to identify mutations in KRAS codons 12 and 13 in agreement with earlier studies.90 Although comparing differences in the limits of detection and quantitation between KRAS tests is a significant consideration for the pathologist in selecting a KRAS testing platform, it is important to note that it is currently not known what level of sensitivity is required to provide useful information to clinicians. Indeed, pathologists must be aware that assays with exceptionally low detection limits may identify patients with mutant KRAS expression in only a limited proportion of tumor tissue.
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
GUIDELINES FOR KRAS MUTATION TESTING
Assessment of KRAS mutational status will likely become a routine aspect of analysis of colorectal cancer, similar to routine HER2 protein analysis by immunohistochemistry or HER2 gene analysis by in situ hybridization in breast cancer. As with HER2/HER2 analysis,95 there is a need for rigorous application of best practices to ensure accurate assessments of KRAS status. There are several ways in which pathologists can play a central role in this process (Fig. 3).89 Pathologists verify the presence of tumor material in the tissue section or block to be used for DNA extraction and assess whether there is sufficient quantity and quality of tumor material for KRAS testing89; this is an especially important function given the observation that KRAS mutations are visble early in tumorigenesis.61,66–68 Pathologists also determine whether the percentage of tumor tissue in the selected tissue section or block is above the lower limit of quantitation of the selected KRAS test used in the laboratory. In addition, pathologists evaluate and select a molecular diagnostic laboratory for KRAS testing, select the appropriate technology for diagnostic laboratories, and guide oncologists and team members in the interpretation of results.89
Two different sets of recommendations for KRAS mutational analysis have recently been proposed by the College of American Pathologists (CAP)89 and European Society of Pathology (ESP).88 These recommendations were generally consistent with one another and emphasized the importance of pathologists in the selection and preparation of tissue samples, KRAS assay selection, and standardized reporting of results.88,89 Although the recommendations by CAP did not include guidelines on the selection of appropriate tumor samples,89 ESP guidelines recommended testing of primary tumor tissue, and if possible, also metastatic tumor, but suggest that adenoma tissue and noninvasive carcinomas not be used for KRAS testing.88 However, because KRAS mutations occur relatively early during colorectal tumor development and frequently remain intact in metastatic tissues,72 sampling of multiple tumor sites (primary and metastatic) from patients with advanced disease may not be absolutely necessary for meaningful KRAS testing results. In contrast, as tumors may contain considerable genetic heterogeneity,80,81 limiting testing for gene mutations to only 1 tissue block could potentially result in failure to identify mutations in KRAS or other genes. Additional investigation is therefore necessary to guide pathologists in the selection of appropriate samples for testing. Microdissection of tumor tissues from FFPE specimens followed by DNA extraction and PCR may offer an option because it allows for the identification of multiple neoplastic subclones within a tumor.81
Regarding the selection of a diagnostic test for KRAS mutations, CAP guidelines state that laboratory-developed tests should be used only if the laboratory has carried out required validation testing and received accreditation by CAP or a similar agency.89 Under Clinical Laboratory Improvement Amendments guidelines, laboratory-developed tests should be validated for these 6 parameters: accuracy, precision, reportable range, reference range, analytical sensitivity, and analytical specificity.96 ESP guidelines recommend that laboratories develop standardized operating procedures and testing requirements.88 Specific recommendations for several testing parameters were made by ESP, including sensitivity, specificity, method validation, analysis success rate, and costs. Specifically, ESP guidelines recommended that the KRAS mutation test selected for use should be capable of detecting at least 7 common mutations in KRAS codons 12 and 13. In addition, the ESP panel proposed the initiation of a European quality-assurance program to ensure that KRAS analyses are as accurate as possible. The CAP Perspectives on Emerging Technology report offered no specific recommendations on reporting of KRAS analyses,89 whereas the ESP guidelines stated that reporting should adhere to the Guidelines for Quality Assurance in Molecular Genetic Testing put forth by the Organization for Economic Cooperation and Development and should include information regarding patient identity, tissue tumor content, tumor type, selected testing method, and KRAS test results and interpretation.88 A recent publication by Monzon et al evaluating the role of KRAS mutation testing in mCRC has also provided recommendations consistent with those described above for reporting KRAS test results.79KRAS proficiency testing is currently offered twice a year by CAP.
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.
QUESTIONS AND FUTURE PERSPECTIVES
The recent guidelines for KRAS mutation testing in the United States and European Union and the development of various assays for KRAS mutation testing raise some important questions for pathologists and the oncology community. How should pathologists address the possibility of mutations in other codons (eg, codon 61) or mutations in codons 12 or 13 that are not detected by many assays? As mutations in codon 61 of KRAS are relatively rare compared with mutations in codons 12 and 13,12,55 many laboratory and commercial diagnostic tests do not screen for them. Detection of KRAS codon 61 mutations can be achieved by pyrosequencing,90 including the commercially available test announced by Qiagen that includes codon 61 in its panel of screened mutations. In general, it is more difficult to assess the association of codon 61 mutation with prognosis and/or response to therapy because this alteration occurs much less frequently, and it is therefore difficult to assess the relevance of this mutation in individual clinical studies. A recent study showed that KRAS mutations in codons 61 and 146 are significantly associated with shorter progression-free survival compared with wild-type KRAS (3.8 vs. 5.1 mo, respectively; P=0.028) in patients with mCRC treated with a combination of cetuximab and chemotherapy.97 Although additional studies with larger numbers of patients will be necessary to confirm the role of these KRAS mutations in the response to EGFR inhibitors, these results suggest that mutations in codons other than 12 and 13 can predict response to anti-EGFR therapy. Consequently, the ability to detect mutations in codons other than 12 and 13 may be necessary to avoid unnecessary use of anti-EGFR therapies in some patients.
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.
KRAS has recently emerged as an important biomarker in mCRC. The presence of mutations in KRAS can predict poor response to therapy with anti-EGFR monoclonal antibodies. KRAS mutational analysis is likely to become an important part of pathology screening for patients with mCRC. A commercial test (TheraScreen) for common mutations in codons 12 and 13 of KRAS has been approved in the European Union for patients with mCRC who are candidates for anti-EGFR therapy. Recommendations for similar pretreatment diagnostics to accompany anti-EGFR therapy in mCRC have been made in the United States. It will be important for pathology laboratories to ensure that patients are screened for KRAS mutations and that the KRAS mutational analysis techniques used provide an accurate assessment of KRAS status. The development and adoption of guidelines for KRAS mutation testing are crucial for success.
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.
1. Green MR. Targeting targeted therapy. N Engl J Med. 2004;350:2191–2193.
2. Van Cutsem E, Peeters M, Siena S, et al. Open-label phase III trial of panitumumab plus best supportive care compared with best supportive care alone in patients with chemotherapy-refractory metastatic colorectal cancer. J Clin Oncol. 2007;25:1658–1664.
3. Jonker DJ, O'Callaghan CJ, Karapetis CS, et al. Cetuximab for the treatment of colorectal cancer. N Engl J Med. 2007;357:2040–2048.
4. Amado RG, Wolf M, Peeters M, et al. Wild-type KRAS
is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol. 2008;26:1626–1634.
5. Lievre A, Bachet JB, Boige V, 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.
6. Benvenuti S, Sartore-Bianchi A, Di Nicolantonio F, et al. 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. 2007;67:2643–2648.
7. Khambata-Ford S, Garrett CR, Meropol NJ, et al. Expression of epiregulin and amphiregulin and K-ras mutation status predict disease control in metastatic colorectal cancer patients treated with cetuximab. J Clin Oncol. 2007;25:3230–3237.
8. Di Fiore F, Van Cutsem E, Laurent-Puig P, et al. Role of KRAS
mutation in predicting response, progression-free survival, and overall survival in irinotecan-refractory patients treated with cetuximab plus irinotecan for a metastatic colorectal cancer: analysis of 281 individual data from published series [abstract]. J Clin Oncol. 2008;26:4035.
9. Cervantes T, Macarulla T, Martinelli E, et al. Correlation of KRAS status [wild type (wt) vs. mutant (mt)] with efficacy to first-line cetuximab in a study of cetuximab single agent followed by cetuximab+FOLFIRI in patients (pts) with metastatic colorectal cancer (mCRC) [abstract]. J Clin Oncol. 2008;26:4129.
10. Finocchiaro G, Cappuzzo F, Jänne PA, et al. EGFR, HER2 and Kras as predictive factors for cetuximab sensitivity in colorectal cancer [abstract]. J Clin Oncol. 2007;25(18S):4021.
11. Tejpar S, Peeters M, Humblet Y, et al. Relationship of efficacy with KRAS 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) [abstract]. J Clin Oncol. 2008;26:4001.
12. Freeman DJ, Juan T, Reiner M, et al. Association of K-ras mutational status and clinical outcomes in patients with metastatic colorectal cancer receiving panitumumab alone. Clin Colorectal Cancer. 2008;7:184–190.
13. Moroni M, Veronese S, Benvenuti S, et al. Gene copy number for epidermal growth factor receptor (EGFR) and clinical response to antiEGFR treatment in colorectal cancer: a cohort study. Lancet Oncol. 2005;6:279–286.
14. Frattini M, Saletti P, Romagnani E, et al. PTEN loss of expression predicts cetuximab efficacy in metastatic colorectal cancer patients. Br J Cancer. 2007;97:1139–1145.
15. Lievre A, Bachet JB, Le Corre D, et al. KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer Res. 2006;66:3992–3995.
16. Di Fiore F, Blanchard F, Charbonnier F, et al. Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by cetuximab plus chemotherapy. Br J Cancer. 2007;96:1166–1169.
17. Karapetis CS, Khambata-Ford S, Jonker DJ, et al. K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med. 2008;359:1757–1765.
18. Bokemeyer C, Bondarenko I, Makhson A, et al. Fluorouracil, leucovorin, and oxaliplatin with and without cetuximab in the first-line treatment of metastatic colorectal cancer. J Clin Oncol. 2009;27:663–671.
19. Van Cutsem E, Kohne CH, Hitre E, et al. Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer. N Engl J Med. 2009;360:1408–1417.
20. Ciardiello F, Tortora G. EGFR antagonists in cancer treatment. N Engl J Med. 2008;358:1160–1174.
21. Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol. 2001;2:127–137.
22. Tzahar E, Waterman H, Chen X, et al. A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor. Mol Cell Biol. 1996;16:5276–5287.
23. Arcaro A, Zvelebil MJ, Wallasch C, et al. Class II phosphoinositide 3-kinases are downstream targets of activated polypeptide growth factor receptors. Mol Cell Biol. 2000;20:3817–3830.
24. Coffer PJ, Kruijer W. EGF receptor deletions define a region specifically mediating STAT transcription factor activation. Biochem Biophys Res Commun. 1995;210:74–81.
25. Karihaloo A, O'Rourke DA, Nickel C, et al. Differential MAPK pathways utilized for HGF- and EGF-dependent renal epithelial morphogenesis. J Biol Chem. 2001;276:9166–9173.
26. Giehl K, Skripczynski B, Mansard A, et al. Growth factor-dependent activation of the Ras-Raf-MEK-MAPK pathway in the human pancreatic carcinoma cell line PANC-1 carrying activated K-ras: implications for cell proliferation and cell migration. Oncogene. 2000;19:2930–2942.
27. Goldstein NS, Armin M. Epidermal growth factor receptor immunohistochemical reactivity in patients with American Joint Committee on Cancer Stage IV colon adenocarcinoma: implications for a standardized scoring system. Cancer. 2001;92:1331–1346.
28. Messa C, Russo F, Caruso MG, et al. EGF, TGF-alpha, and EGF-R in human colorectal adenocarcinoma. Acta Oncol. 1998;37:285–289.
29. Tsao MS, Sakurada A, Cutz JC, et al. Erlotinib in lung cancer-molecular and clinical predictors of outcome. N Engl J Med. 2005;353:133–144.
30. Rusch V, Baselga J, Cordon-Cardo C, et al. Differential expression of the epidermal growth factor receptor and its ligands in primary non-small cell lung cancers and adjacent benign lung. Cancer Res. 1993;53(10 suppl):2379–2385.
31. Vectibix® (panitumumab). Full Prescribing Information. Thousand Oaks, CA: Amgen Inc.; 2009.
32. Vectibix European Public Assessment Report, Scientific Discussion
. London, UK: European Medicines Agency; 2008.
33. Vectibix® (panitumumab). Full Prescribing Information. Mississauga, ON, Canada: Amgen Inc.; 2008.
34. Erbitux® (cetuximab). Full Prescribing Information. Princeton, NJ: Bristol-Myers Squibb Company; 2009.
35. Erbitux European Public Assessment Report, Scientific Discussion.
London, UK: European Medicines Agency; 2009.
36. Grunwald V, Hidalgo M. Developing inhibitors of the epidermal growth factor receptor for cancer treatment. J Natl Cancer Inst. 2003;95:851–867.
37. Tarceva® (erlotinib). Full Prescribing Information. Melville, NY: OSI Pharmaceuticals, Inc.; 2008.
38. Ellis RW, DeFeo D, Furth ME, et al. Mouse cells contain two distinct ras gene mRNA species that can be translated into a p21 onc protein. Mol Cell Biol. 1982;2:1339–1345.
39. Malumbres M, Barbacid M. RAS oncogenes: the first 30 years. Nat Rev Cancer. 2003;3:459–465.
40. Yamashita T, Yamamoto K, Kikuchi A, et al. Purification and characterization of c-Ki-ras p21 from bovine brain crude membranes. J Biol Chem. 1988;263:17181–17188.
41. Der CJ, Krontiris TG, Cooper GM. Transforming genes of human bladder and lung carcinoma cell lines are homologous to the ras genes of Harvey and Kirsten sarcoma viruses. Proc Natl Acad Sci U S A. 1982;79:3637–3640.
42. Takai Y, Sasaki T, Matozaki T. Small GTP-binding proteins. Physiol Rev. 2001;81:153–208.
43. Buday L, Downward J. Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor. Cell. 1993;73:611–620.
44. Downward J. Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer. 2003;3:11–22.
45. Gibbs JB, Schaber MD, Allard WJ, et al. Purification of ras GTPase activating protein from bovine brain. Proc Natl Acad Sci U S A. 1988;85:5026–5030.
46. Sharpe CC, Dockrell ME, Scott R, et al. Evidence of a role for Ki-Ras in the stimulated proliferation of renal fibroblasts. J Am Soc Nephrol. 1999;10:1186–1192.
47. Clyde-Smith J, Silins G, Gartside M, et al. Characterization of RasGRP2, a plasma membrane-targeted, dual specificity Ras/Rap exchange factor. J Biol Chem. 2000;275:32260–32267.
48. Jaiswal BS, Janakiraman V, Kljavin NM, et al. Combined targeting of BRAF and CRAF or BRAF and PI3K effector pathways is required for efficacy in NRAS mutant tumors. PLoS One. 2009;4:e5717.
49. Tchernitsa OI, Sers C, Zuber J, et al. Transcriptional basis of KRAS oncogene-mediated cellular transformation in ovarian epithelial cells. Oncogene. 2004;23:4536–4555.
50. Pohl G, Ho CL, Kurman RJ, et al. Inactivation of the mitogen-activated protein kinase pathway as a potential target-based therapy in ovarian serous tumors with KRAS or BRAF mutations. Cancer Res. 2005;65:1994–2000.
51. Tang Y, Zhou H, Chen A, et al. The Akt proto-oncogene links Ras to Pak and cell survival signals. J Biol Chem. 2000;275:9106–9109.
53. Schubbert S, Shannon K, Bollag G. Hyperactive Ras in developmental disorders and cancer. Nat Rev Cancer. 2007;7:295–308.
54. Scheffzek K, Ahmadian MR, Kabsch W, et al. The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science. 1997;277:333–338.
55. Lee J, Jang KT, Ki CS, et al. Impact of epidermal growth factor receptor (EGFR) kinase mutations, EGFR gene amplifications, and KRAS mutations on survival of pancreatic adenocarcinoma. Cancer. 2007;109:1561–1569.
56. Garcia-Rostan G, Zhao H, Camp RL, et al. Ras mutations are associated with aggressive tumor phenotypes and poor prognosis in thyroid cancer. J Clin Oncol. 2003;21:3226–3235.
57. Eberhard DA, Johnson BE, Amler LC, et al. Mutations in the epidermal growth factor receptor and in KRAS are predictive and prognostic indicators in patients with non-small-cell lung cancer treated with chemotherapy alone and in combination with erlotinib. J Clin Oncol. 2005;23:5900–5909.
58. Andreyev HJ, Norman AR, Cunningham D, et al. Kirsten ras mutations in patients with colorectal cancer: the ‘RASCAL II’ study. Br J Cancer. 2001;85:692–696.
59. Andreyev HJ, Norman AR, Cunningham D, et al. Kirsten ras mutations in patients with colorectal cancer: the multicenter “RASCAL” study. J Natl Cancer Inst. 1998;90:675–684.
61. Vogelstein B, Fearon ER, Hamilton SR, et al. Genetic alterations during colorectal-tumor development. N Engl J Med. 1988;319:525–532.
62. Esteller M, Gonzalez S, Risques RA, et al. K-ras and p16 aberrations confer poor prognosis in human colorectal cancer. J Clin Oncol. 2001;19:299–304.
63. Bazan V, Migliavacca M, Zanna I, et al. Specific codon 13 K-ras mutations are predictive of clinical outcome in colorectal cancer patients, whereas codon 12 K-ras mutations are associated with mucinous histotype. Ann Oncol. 2002;13:1438–1446.
64. Ince WL, Jubb AM, Holden SN, et al. Association of k-ras, b-raf, and p53 status with the treatment effect of bevacizumab. J Natl Cancer Inst. 2005;97:981–989.
65. Bouzourene H, Gervaz P, Cerottini JP, et al. p53 and Ki-ras as prognostic factors for Dukes' stage B colorectal cancer. Eur J Cancer. 2000;36:1008–1015.
66. Jen J, Powell SM, Papadopoulos N, et al. Molecular determinants of dysplasia in colorectal lesions. Cancer Res. 1994;54:5523–5526.
67. Forrester K, Almoguera C, Han K, et al. Detection of high incidence of K-ras oncogenes during human colon tumorigenesis. Nature. 1987;327:298–303.
68. Bos JL, Fearon ER, Hamilton SR, et al. Prevalence of ras gene mutations in human colorectal cancers. Nature. 1987;327:293–297.
69. Pretlow TP, Brasitus TA, Fulton NC, et al. K-ras mutations in putative preneoplastic lesions in human colon. J Natl Cancer Inst. 1993;85:2004–2007.
70. Etienne-Grimaldi MC, Formento JL, Francoual M, et al. K-Ras mutations and treatment outcome in colorectal cancer patients receiving exclusive fluoropyrimidine therapy. Clin Cancer Res. 2008;14:4830–4835.
71. Artale S, Sartore-Bianchi A, Veronese SM, et al. Mutations of KRAS and BRAF in primary and matched metastatic sites of colorectal cancer. J Clin Oncol. 2008;26:4217–4219.
72. Losi L, Benhattar J, Costa J. Stability of K-ras mutations throughout the natural history of human colorectal cancer. Eur J Cancer. 1992;28A:1115–1120.
73. Schimanski CC, Linnemann U, Berger MR. Sensitive detection of K-ras mutations augments diagnosis of colorectal cancer metastases in the liver. Cancer Res. 1999;59:5169–5175.
74. Oudejans JJ, Slebos RJ, Zoetmulder FA, et al. Differential activation of ras genes by point mutation in human colon cancer with metastases to either lung or liver. Int J Cancer. 1991;49:875–879.
75. Albanese I, Scibetta AG, Migliavacca M, et al. Heterogeneity within and between primary colorectal carcinomas and matched metastases as revealed by analysis of Ki-ras and p53 mutations. Biochem Biophys Res Commun. 2004;325:784–791.
76. Suchy B, Zietz C, Rabes HM. K-ras point mutations in human colorectal carcinomas: relation to aneuploidy and metastasis. Int J Cancer. 1992;52:30–33.
77. Al-Mulla F, Going JJ, Sowden THH, et al. Heterogeneity of mutant versus wild-type Ki-ras in primary and metastatic colorectal carcinomas, and association of codon-12 valine with early mortality. J Pathol. 1998;185:130–138.
78. Santini D, Loupakis F, Vincenzi B, et al. High concordance of KRAS status between primary colorectal tumors and related metastatic sites: implications for clinical practice. Oncologist. 2008;13:1270–1275.
79. Monzon FA, Ogino S, Hammond ME, et al. The role of KRAS mutation testing in the management of patients with metastatic colorectal cancer. Arch Pathol Lab Med. 2009;133:1600–1606.
80. Losi L, Baisse B, Bouzourene H, et al. Evolution of intratumoral genetic heterogeneity during colorectal cancer progression. Carcinogenesis. 2005;26:916–922.
81. Baisse B, Bouzourene H, Saraga EP, et al. Intratumor genetic heterogeneity in advanced human colorectal adenocarcinoma. Int J Cancer. 2001;93:346–352.
82. Amado RG, Wolf M, Freeman D, et al. Association of KRAS
mutational status and efficacy of panitumumab monotherapy for the treatment (tx) of metastatic colorectal cancer (mcrc): results of pooled data from 4 clinical studies [abstract]. Ann Oncol. 2008;19(S8):viii126.
83. Miller VA, Riely GJ, Zakowski MF, et al. Molecular characteristics of bronchioloalveolar carcinoma and adenocarcinoma, bronchioloalveolar carcinoma subtype, predict response to erlotinib. J Clin Oncol. 2008;26:1472–1478.
84. Mittmann N, Au HJ, Tu D, et al. Prospective cost-effectiveness analysis of cetuximab in metastatic colorectal cancer: evaluation of National Cancer Institute of Canada Clinical Trials Group CO.17 trial. J Natl Cancer Inst. 2009;101:1182–1192.
85. Allegra CJ, Jessup JM, Somerfield MR, et al. American Society of Clinical Oncology provisional clinical opinion: testing for KRAS gene mutations in patients with metastatic colorectal carcinoma to predict response to anti-epidermal growth factor receptor monoclonal antibody therapy. J Clin Oncol. 2009;27:2091–2096.
86. National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology: Colon Cancer v.2.2009. Fort Washington, PA: National Comprehensive Cancer Network; 2009.
87. National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology: Rectal Cancer v.2.2009. Fort Washington, PA: National Comprehensive Cancer Network; 2009.
88. van Krieken JH, Jung A, Kirchner T, et al. KRAS mutation testing for predicting response to anti-EGFR therapy for colorectal carcinoma: proposal for an European quality assurance program. Virchows Arch. 2008;453:417–431.
89. Perspectives on Emerging Technology (POET). Report: KRAS Mutation Testing for Colorectal Cancer (CRC). Northfield, IL: College of American Pathologists; 2009.
90. Poehlmann A, Kuester D, Meyer F, et al. K-ras mutation detection in colorectal cancer using the Pyrosequencing technique. Pathol Res Pract. 2007;203:489–497.
91. Voelkerding KV, Dames SA, Durtschi JD. Next-generation sequencing: from basic research to diagnostics. Clin Chem. 2009;55:641–658.
92. Juan T, Suggs S, Wolf M, et al. A comparability study of 4 commercial KRAS tests. Presented at: Annual Meeting of the American Association of Cancer Researchers; April 12-14, 2008; San Diego, CA.
93. Whitehall V, Tran K, Umapathy A, et al. A multicenter blinded study to evaluate KRAS
mutation testing methodologies in the clinical setting. J Mol Diagn. 2009;11:543–552.
94. Tol J, Dijkstra JR, Vink-Borger ME, et al. High sensitivity of both sequencing and real-time PCR analysis of KRAS mutations in colorectal cancer tissue. J Cell Mol Med. 2009; [Epub ahead of print].
95. Wolff AC, Hammond ME, Schwartz JN, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol. 2007;25:118–145.
96. Medicare, medicaid, and CLIA programs; laboratory requirements relating to quality systems and certain personnel qualifications; final rule. Vol 68: U.S. Department of Health and Human Services Federal Register; 2003:3640–3714.
97. Loupakis F, Ruzzo A, Cremolini C, et al. KRAS codon 61, 146 and BRAF mutations predict resistance to cetuximab plus irinotecan in KRAS codon 12 and 13 wild-type metastatic colorectal cancer. Br J Cancer. 2009;101:715–721.
98. Freeman D, Amado R, Wolf M, et al. Efficacy of panitumumab monotherapy in relation to KRAS
mutational status for treating metastatic colorectal cancer (mCRC) from four clinical studies [abstract]. Presented at: ASCO-NCI-EORTC Annual Meeting on Molecular Markers in Cancer; October 30-November 1, 2008; Hollywood, FL.
99. Di Nicolantonio F, Martini M, Molinari F, et al. Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer. J Clin Oncol. 2008;26:5705–5712.
100. Sartore-Bianchi A, Martini M, Molinari F, et al. PIK3CA mutations in colorectal cancer are associated with clinical resistance to EGFR-targeted monoclonal antibodies. Cancer Res. 2009;69:1851–1857.
This article has been cited 8 time(s).
Drug Development ResearchIssues in Omics Data Integration for Gene Set Analysis and Aberrant Pathway IdentificationDrug Development Research
American Journal of SurgeryCSE1L modulates Ras-induced cancer cell invasion: correlation of K-Ras mutation and CSE1L expression in colorectal cancer progressionAmerican Journal of Surgery
Diagnostic PathologyA comparability study of 5 commercial KRAS testsDiagnostic Pathology
Future OncologyRecently identified and potential targets for colon cancer treatmentFuture Oncology
Cancer Treatment ReviewsEffect of inhibition of the FGFR-MAPK signaling pathway on the development of ocular toxicitiesCancer Treatment Reviews
Genetics in MedicineRecommendations from the EGAPP Working Group: can testing of tumor tissue for mutations in EGFR pathway downstream effector genes in patients with metastatic colorectal cancer improve health outcomes by guiding decisions regarding anti-EGFR therapy?Genetics in Medicine
Journal of Clinical PathologyGene of the month: KRASJournal of Clinical Pathology
HistopathologyKRAS mutation is present in a small subset of primary urinary bladder adenocarcinomasHistopathology
KRAS; metastatic colorectal cancer; epidermal growth factor receptor; panitumumab; cetuximab
© 2010 Lippincott Williams & Wilkins, Inc.
Highlight selected keywords in the article text.