Curr Oncol Rep (2010) 12:160–167 DOI 10.1007/s11912-010-0099-y

KRAS Testing and Its Importance in Colorectal Cancer Deepa T. Patil & Cory R. Fraser & Thomas P. Plesec

Published online: 30 March 2010 # Springer Science+Business Media, LLC 2010

Abstract Cetuximab and panitumumab are monoclonal antibodies used in the treatment of metastatic colorectal cancer (mCRC) by selectively targeting the epidermal growth factor receptor (EGFR) axis. Studies have shown that mutations in codons 12/13 of exon 2 of the KRAS gene render these therapies ineffective. As a result, the National Comprehensive Cancer Network and American Society of Clinical Oncology recommend KRAS mutation testing in mCRC. Appropriate testing depends on the coordinated efforts of the entire treatment team, including the pathologist, who selects the tumor sample and testing platform as well as interprets and reports results. In addition to describing rationale and methodologies for KRAS mutation testing, the authors also summarize their algorithmic approach and elaborate the potential role of newer molecular biomarkers to predict anti-EGFR resistance in wild-type KRAS tumors.

only hope for cure in colorectal cancer, but approximately 20% of patients present with stage IV disease. New chemotherapeutic options, notably irinotecan, oxaliplatin, bevacizumab, and capecitabine, in addition to efficient screening programs, improved surgical techniques, and risk factor modifications, have led to decreased colorectal cancer-related mortality. Nevertheless, about 50% of patients will have distant metastases during their disease course, and cetuximab and panitumumab are biologics used in the management of metastatic disease [2]. These agents are monoclonal antibodies that target the epidermal growth factor receptor (EGFR). EGFRs are overexpressed in numerous cancers and are associated with a poor prognosis in most studies. Following the discovery of its tyrosine kinase activity and its homology with v-erb-b, a viral oncogene, several experiments showed that over expression of EGFR can be a transforming event in carcinogenesis [3].

Keywords KRAS . Mutation . Colorectal cancer . Codon 12 . Codon 13 . Cetuximab . Panitumumab EGFR and Colorectal Cancer Introduction An estimated 146,970 new cases of colorectal cancer and 49,920 deaths occurred in 2009 [1]. Surgery remains the

D. T. Patil : C. R. Fraser : T. P. Plesec (*) Department of Anatomic Pathology, Cleveland Clinic, L25, 9500 Euclid Avenue, Cleveland, OH 44195, USA e-mail: [email protected] D. T. Patil e-mail: [email protected] C. R. Fraser e-mail: [email protected]

EGFR is a 170-kDa transmembrane tyrosine kinase receptor that is present in most epithelial tissues. It belongs to the erBb/HER family of cell surface receptors, which consists of four structurally related proteins. Activation of the EGFR signaling pathway occurs when ligands such as EGF, transforming growth factor-α, amphiregulin, epiregulin, and others [4] bind to the extracellular domain, leading to homodomerization or heterodimerization. This triggers autophosphorylation of the intracellular tyrosine kinase residues, inducing activation of multiple signal transduction pathways, including the RAS/MAPK, PI3K/AKT, and STAT/AKT. These pathways play critical roles in cell cycle progression, cell proliferation and survival, adhesion, angiogenesis, migration, and invasion, all of which are

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key components of tumor pathogenesis [5]. EGFR is overexpressed in up to 82% [6] of colorectal cancers and has been associated with advanced-stage disease [7]. Increased expression by immunohistochemistry (IHC) has been documented in the deepest portions of tumors, implicating its role in carcinoma progression [8]. Cetuximab is a human–mouse chimeric immunoglobulin G1 monoclonal antibody that binds exclusively to the extracellular domain of EGFR, leading to inhibition of EGFR-mediated cell signaling [9]. It was approved by the US Food and Drug Administration (FDA) in 2004 as a second-line treatment of metastatic colorectal cancer (mCRC) [10]. Panitumumab, the first fully humanized immunoglobulin G2 monoclonal antibody, was FDA approved in 2007 for treatment of refractory mCRC [9]. Unfortunately, clinical trials have demonstrated that only 8% to 23% of unselected patients with metastases respond to these antibodies [6, 11]. Moreover, these therapies are extremely expensive, costing approximately $70,000/patient and have side effects, most commonly an acne-like rash. Due to the expenses, potential toxicities, and relatively small proportion of anti-EGFR “responders,” much investigation has been directed at identifying biomarkers to predict clinical response. EGFR protein expression by IHC was the original method of patient selection for anti-EGFR therapy ever since the pivotal BOND trial; however, the similar response rates in EGFR-positive and EFGR-negative tumors has excluded EGFR analysis by IHC as a selection criterion for anti-EGFR therapies [12]. In addition, less than 1% of colorectal cancers exhibit EGFR tyrosine kinase mutations; therefore EGFR gene mutations do not play a role in predicting anti-EGFR therapy response. The failure of EGFR by IHC or mutational analysis as a predictive biomarker led to the discovery of the utility of KRAS.

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activity or insensitivity to GAPs [15]. Single nucleotide point mutations are generally reported in 30% to 40% of colorectal cancers. Activating mutations are most common in codons 12 and 13 (exon 2) of the KRAS gene (Fig. 1), with the majority (∼80%) occurring in codon 12 [16]. Activating mutations in codons 61, 146, and 154 have also been reported, but these account for less than 10% of mutations [10]. Multiple clinical trials have confirmed that a subset of patients with KRAS wild-type tumors may benefit from cetuximab or panitumumab therapy, whereas those harboring KRAS exon 2 codon 12/13 mutations almost universally fail anti-EGFR therapy [17–19•, 20]. The survival benefit, if found at all, is measured only in months. The response rates for anti-EGFR therapy in relation to KRAS mutational status are summarized in Table 1. In a retrospective analysis of 100 patients treated with cetuximab as second or subsequent-line therapy, none of the 32 patients with a KRAS-mutant tumor responded to treatment [21]. In another multicenter retrospective analysis, KRAS mutation was significantly associated with resistance to response in all 24 KRAS-mutated tumors, compared to a 40% response rate in KRAS wild-type tumors [20]. The CRYSTAL trial found no significant difference in progression-free survival (PFS) or overall survival (OS) among those treated with cetuximab + FOLFIRI, regardless of KRAS mutation status; however, there was a significantly improved response rate in KRAS wild-type tumors (59.3%) compared to mutant KRAS tumors (36.2%) [22]. On the contrary, the CO.17 trial showed significantly improved OS and PFS in patients with KRAS wild-type tumors treated with cetuximab compared with supportive care alone. No such difference was observed in patients with mutated KRAS tumors [23••]. Similarly, in the OPUS trial, addition of cetuximab to FOLFOX-4 regimen was associated with a 43% reduction in risk of progression in patients with KRAS wild-type

KRAS: Role as Biomarker for Anti-EGFR Response KRAS, the human homolog of the Kirsten rat sarcoma-2 virus oncogene, encodes a small G-protein that functions downstream of EGFR. RAS proteins cycle between an active GTP-bound phase and an inactive GDP-bound phase. This is promoted by guanine exchange factors (GEFs) that mediate the GDP–GTP exchange. Hydrolysis of the active (GTP-bound) to inactive (GDP-bound) phase is mediated by GTPase-activating proteins (GAPs). EGFR activation promotes the activity of GEFs, in turn activating KRAS and the RAS/MAPK pathway, which helps to regulate apoptosis, cell growth, adhesion, and differentiation [13, 14]. Activating KRAS mutations result in a permanently active GTP-bound form due to inhibition of the GTPase

G

C

T

G 113

K

T

G

G 117

C

G

T

Fig. 1 KRAS forward sequencing electropherogram depicting point mutations in exon 2 codons 12 (left arrow) and 13 (right arrow) of the KRAS gene. Each mutant peak underlies the normal base peak

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Table 1 Biomarkers of anti-EGFR therapy response

KRAS codon 12/13

KRAS codon 61 BRAF

PIK3CA

PTEN

Mutation frequency, %

Wild-type/normal expression response rate, %

Mutant/loss of expression response rate, %a

42 [24••] 30 [19•] 43 [25•] 42 [23••] 36 [22] 27 [20] 37 [21] 27 [47] 8 [38]

33 6 0 1 36 0 0 LOR 0

14 [19•] 15 [38]

60 28 17 13 59 40 23 – 32 (includes codon 146) 32 32

0 0

8 [21] 8 [47] 13 [40] 14 [39] 12 [47] 15 [21] 12 [48] 7 [49] 10 [41] 20 [45] 14 [44] 41 [42] 36 [47] 13 [40]

17 NS – NR NS – 30 – – – 42 63 – NR

0 NS LOR 0 NS NS 36 NS NS NS 13 0 LOR 0

EGFR epidermal growth factor receptor; LOR lack of objective response; NR not reported; NS not significant in terms of response; PTEN phosphatase and tensin homolog a Patients who responded with KRAS codon 12/13 mutation or PTEN loss were generally treated with conventional chemotherapy in addition to antiEGFR therapy

tumors [24••]. While most studies have investigated cetuximab, a significant survival benefit also was observed in patients with KRAS wild-type tumors treated with panitumumab, in a prospective trial of 427 metastatic colorectal carcinoma patients [25•]. Selecting patients on the basis of KRAS mutation status is, however, an imperfect process. At least 40% of patients with wild-type KRAS show no response to anti-EGFR therapy [25•]. This indicates the potential for additional biomarkers to better select those who will benefit from antiEGFR therapy. Some of these biomarkers include other KRAS mutations, BRAF, EGFR copy number, PI3K (phosphoinositide 3-kinase), and PTEN (phosphatase and tensin homolog), and these will be discussed later.

Methods of Detection Although National Comprehensive Cancer Network (NCCN) and American Society of Clinical Oncology (ASCO) guidelines recommend KRAS codon 12/13 mutation testing, there is no “official” method of testing. There are no FDA-approved methods currently, but this is expected to change in the near future. The method of choice is laboratory dependent, as there is probably no longer a gold standard. Laboratories must take into account laboratory experience with a particular method, existing laboratory infrastructure, personnel, platform availability, and costs when choosing their KRAS testing platform. Laboratories must be CLIA approved for high-complexity testing and be compliant with the standard quality control and quality assurance methods. To that end, the College of American Pathologists offers biannual KRAS mutation proficiency testing. KRAS mutational testing is complicated because the specimen is invariably composed of both non-neoplastic and neoplastic tissue, and the neoplasm may or may not contain a KRAS mutation. Although mutations are usually heterozygous, homozygous or compound heterozygous mutations do occur. A heterozygous mutation occurring in a background of abundant non-tumor tissue is significantly diluted by wild-type DNA that may impair the detection of a mutation; in addition, rare mutation variants may be missed by some methods. The techniques used to detect KRAS mutations vary in their analytic sensitivity and throughput and raise issues for both the clinician and laboratory. There are seemingly endless methods reported in the literature that may be employed in detecting KRAS mutations. The major methods include direct DNA sequencing (ie, Sanger method), allele-specific polymerase chain reaction (PCR), high-resolution melt curve analysis, pyrosequencing, and restriction fragment length polymorphism; however, numerous other methods are being explored. A common feature of these major methods is the use of nucleic acid amplification (eg, PCR). Among the various methodologies, the Sanger method, melt curve analysis, and allele-specific PCR hold particular significance because these were employed in the KRAS mutation analyses of the cetuximab and panitumumab clinical trials. The Sanger method is often referred to as the “gold standard” because of the extensive experience with this method, its capability to provide the specific point mutation, and its ability to detect all possible mutations. Drawbacks include low sensitivity (requires at least 20% mutant cells) and long turnaround time, often ranging from 4 days to 2 weeks [26]. The turnaround time can be delayed further due to technical difficulties or poor-quality DNA. If technical difficulties arise, we have found that re-testing a

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separate tumor block, if available, often yields satisfactory results. Allele-specific PCR has greater sensitivity (<10% mutant cells, perhaps as low as 1%) compared to direct sequencing, along with the added benefit of a shorter turnaround time, which can be less than 2 days [26]. The assay requires sequence-specific probes and will only identify those mutations that are targeted; some assays do not contain probes for all possible KRAS codon 12/13 mutations. These allele-specific probes allow for selective DNA amplification of only mutant alleles, which is the source of the impressive sensitivity. Some methods also include “blocker” substances to prevent amplification of background wild-type DNA, further reducing the dilutional effect of normal DNA. Many allele-specific assays are also available as commercially available kits, which, although more expensive, provide convenience and potentially greater standardization than assays developed in laboratories, so-called “homebrews.” High-resolution melt curve analysis is based on the principle that sequences, which have undergone mutation, will have less affinity for a wild-type DNA probe and separate at a lower temperature, compared to the unmutated state [26]. This technique has been used to assess KRAS mutational status in colorectal cancer [27]. Though the technology has a reported sensitivity of 5%, the technique’s drawbacks include a long turnaround time (4 days to 2 weeks), which may be prolonged by the need for subsequent confirmatory sequence analysis [26]. Restriction fragment length polymorphism is based on KRAS mutations causing an alteration in cut sites by restriction enzymes when compared to wild type. The technique is exquisitely sensitive (reportedly 0.1%), but is hampered by a turnaround time similar to high-resolution melt curve analysis (4 days to 2 weeks) and also may be prolonged by the need for subsequent sequence analysis in mutation-positive cases [26]. Pyrosequencing is a real-time quantitative assay that detects short stretches of DNA sequences. Its strength is in its ability to detect mutations that occur in clusters, making it well-suited for KRAS mutation testing [28]. The assay is reportedly quite sensitive, efficient, and flexible, as it affords the ability to run separate assays on the same run [29]. This may be a considerable asset in the future should “EGFR panels” come to light (see below). Until recently, there was little in terms of comparative analysis of methodologies and the ability to predict those individuals with “significant” KRAS mutations. Recently, separate publications and abstracts comparing various aspects of KRAS testing have come to light [26, 30•]. Weichert et al. [30•] compared Sanger sequencing, array analysis, melt curve analysis, and pyrosequencing and determined that they yielded similar results. The compar-

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ative analysis also looked at the laboratory testing time requirements, equipment costs, and cost per test. Their findings help to reinforce that there is no longer a gold standard, there are several suitable methods, and the method of choice is laboratory dependent.

Necessary Materials The most commonly used specimens for KRAS mutational analysis are formalin-fixed paraffin-embedded specimens from either the primary or metastatic lesion. Specimen adequacy needs to be validated for each method employed, and the laboratory must be aware of the minimum tumor cell concentration that yields reliable results. This is usually obtained through knowledge of the particular test’s sensitivity as well as performing serial dilutions on tumor cell lines. Our laboratory’s current method of KRAS mutation testing is direct sequencing. Not infrequently, we struggle with determining whether a specimen is adequate for analysis. We routinely perform macro-dissection techniques via meticulous dotting on the hematoxylin and eosin–stained section to isolate the densest area of tumor cells. Then one of our laboratory technicians simply uses a scalpel and a dissecting microscope to scrape off the area of circled tumor to best eliminate non-neoplastic epithelium, stroma, and contaminating inflammatory cells—all of which are cellular elements containing dilutional normal DNA. It remains unclear whether the primary or metastatic tumor should be tested, if both are available. It can be argued that the primary tumor should be tested, since most clinical studies seem to be based on testing of the primary tumor. It is the metastatic disease that prompts anti-EGFR treatment, so it also can be argued that testing the metastasis is the most clinically relevant tissue sample. Furthermore, KRAS mutations are an early event in tumorigenesis and stable throughout the disease process [31], with concordance rates between the primary and metastases generally more than 90% [32]. We generally prefer to test the primary tumor resection because much of the outcome data are based on testing the primary, and, more importantly, the primary resections are much more likely to harbor densely populated areas of tumor. If the primary tumor is unavailable or unsatisfactory, then the metastasis is completely appropriate to test. When testing the primary tumor resection is not possible, testing of core needle biopsies or cytology specimens from metastatic lesions may be required. The pathologist plays a critical role in determining adequacy of these limited samples of tumor as well as locating the area with the densest concentration of tumor. In addition, metastatic lesions tend to have abundant necrosis that far exceeds what was seen in the primary lesion, and tumor necrosis

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occasionally inhibits DNA amplification. Fortunately, this inhibition usually can be overcome by laboratory methods. It has been shown that cytologic specimens from mCRC can provide adequate tissue for KRAS mutational analysis. It also appears that successful mutational analysis can be achieved on both Diff-Quik and Papanicolaou-stained cytospins as well as direct smears [33]. Solomon et al. [34] compared the adequacy of core needle biopsy specimens and the subsequent resection specimens in cases of lung cancer. In their study, 16 of 18 specimens yielded adequate material from both 18-gauge and 20-gauge needles, and the 16 adequate specimens had 100% agreement in KRAS mutation status when compared to the resection specimen. A paucity of data exists on the testing of tumors in patients who have undergone neoadjuvant chemoradiation therapy; however, preliminary data on a study of 18 rectal adenocarcinoma patients showed that KRAS status was unchanged when treated rectal cancers were compared to pre-therapy biopsies, suggesting that post-therapy material is appropriate for testing (Bronner, Personal communication). From our experience, the greatest impact neoadjuvant therapy has on KRAS testing is the significantly reduced tumor:non-tumor ratio. Often, therapy leads to minute islands of tumor buried within variably inflamed and fibrotic tissue. In these cases, we attempt to locate pre-treatment biopsies or metastatic samples. If these cannot be found, we still attempt the mutation testing with very careful macro-dissection. If a wild-type genotype is found, we add a disclaimer that the results were generated from a limited sampling with the possibility of a false wild-type result.

Results Reporting Aside from the usual standard information, a KRAS mutation analysis report should include the type of material used, testing method, and results [35]. Some have argued that the percent of tumor cells within the sample, the analytical sensitivity, and an interpretation of the results also be included [36]. For example, the current ASCO guidelines suggest that reports on patients with KRAS mutations include a statement that treatment with antiEGFR monoclonal antibody therapy is not recommended based on the ASCO provisional clinical opinion [36]. The decision to include the exact KRAS mutation in a report is one of considerable debate. To date, there is no conclusive evidence that different mutations have different prognostic or therapeutic significance, so we think that it is not necessary to include this information within a report. This may change in the future, and maintaining records of the specific mutation for future reference may be warranted, if the laboratory’s testing method allows.

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Our Approach In addition to metastatic disease, some have suggested that KRAS testing be considered at the time of initial diagnosis of stage II and III tumors to avoid any delay in therapy, if it becomes necessary [35]. The NCCN and ASCO recommend testing in individuals with mCRC (stage IV only) who are being considered for treatment with anti-EGFR therapy [2, 36]. On the other end of the spectrum, some laboratories are testing all cancers. We reflexively test all stage III and IV colorectal carcinomas. Stage IV testing complies with current recommendations, and stage III testing enables timely enrollment into anti-EGFR–based clinical trials. As there is no correct algorithm for reflex testing, it is likely that it will remain institution specific, requiring input and an open dialogue between all members of the treatment team. Our reports include the type of material used, testing method, analytical sensitivity, and results including the codon containing the mutation, if applicable. We do not report the specific point mutation (eg, guanine to cytosine), nor do we provide an interpretation of clinical implications, because we are comfortable that our oncologists understand the clinical implications.

Future Since the widespread use of KRAS mutation testing in mCRC, a number of studies have been aimed at identifying nonresponders to anti-EGFR therapy in individuals with KRAS codon 12/13 wild-type tumors. Much like the relationship of KRAS to EGFR, it appears that mutations affecting other genes in the EGFR signaling pathway may also influence response to anti-EGFR therapy in KRAS codon 12/13 wild-type tumors (Table 1). The valine to glutamate mutation at residue 600 within exon 15, induced by the T1799A nucleotide transversion, leads to the vast majority of BRAF mutations in colorectal carcinomas and appears to be an early event in tumorigenesis [37]. In the EGFR signaling pathway, BRAF is downstream from KRAS. Mutations in BRAF and KRAS are mutually exclusive and there are abundant data, second only to KRAS codon 12/13 studies, demonstrating the predictive value of BRAF V600E. Di Nicolantonio et al. [19•] studied 79 cases with wild-type KRAS and found BRAF V600E mutations in 11 (14%). None of the BRAF V600E cases responded to anti-EGFR therapy, versus 32% of KRAS wild-type/BRAF wild-type tumors. In a similar fashion, Loupakis et al. [38] found that compared to 68% of cases with KRAS wild-type/ BRAF wild-type tumors, none of the 13 (0%) KRAS wildtype/BRAF mutant tumors responded to therapy (cetuximab plus irinotecan). Souglakos et al. [21] also noted that none of

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the nine patients in whom the tumor harbored a BRAF mutation responded to cetuximab (plus chemotherapy), while an objective response was noted in 14 of 83 (17%) wild-type BRAF cases. Although not currently recognized by NCCN or ASCO, there appears to be sufficient evidence to warrant exclusion of anti-EGFR therapy in BRAF-mutated tumors. EGFR signaling involves several pathways. On one side, KRAS activates BRAF, while in a parallel fashion, PIK3CA counteracts PTEN and plays a role in the PI3K/ AKT signaling pathway [39]. The PIK3CA gene encodes the p110-α catalytic subunit of PI3K protein. In one study of 110 patients with mCRC, 15 (13.6%) and 32 (29.0%) had PIK3CA and KRAS mutations, respectively [39]. In this study, significant clinical resistance to panitumumab or cetuximab was noted in cases with KRAS wild-type/ PIK3CA-mutated tumors. Perrone et al. [40] also found that PIK3CA mutations correlated with an absence of response to cetuximab plus irinotecan. In contrast, a roughly equal number of studies investigating PIK3CA have shown no significant difference in response to antiEGFR therapies [20, 41]; therefore, the issue of PIK3CA and its role in identifying individuals who will respond to anti-EGFR therapy remains to be resolved. PTEN inhibits the PI3K/AKT signaling pathway and its expression is usually assessed using IHC analysis, but fluorescence in situ hybridization (FISH) has been employed in a minority of studies. In one study, 10 of 16 patients with tumors exhibiting normal PTEN expression by IHC had a partial response to cetuximab, but no response was seen in 11 patients with PTEN-negative tumors [42]. Other studies have yielded similar results [40, 43, 44]; however, Loupakis et al. [38] found that PTEN status assessed using IHC on the primary tumor did not predict response to anti-EGFR therapy. The same study did find that PTEN expression on the metastasis was associated with response when compared to PTEN-negative metastases. Much like PIK3CA, PTEN’s role in identifying patients responsive to anti-EGFR therapy remains to be determined. The significance of other KRAS mutations is also being explored. KRAS codons 61 and 146 are less common activating mutations in the KRAS gene that have, presumably, the same treatment ramifications as KRAS codons 12/13 mutations. In fact, some institutions include codon 61 mutation analyses, despite no official recommendations. Loupakis et al. [38] found that compared with 32% of responders with wild-type KRAS tumors, none of the patients with codon 61/146 mutations (9%) responded to cetuximab plus irinotecan therapy. EGFR gene amplification has also been found to play a role in response to anti-EGFR therapy. It is usually assessed by FISH or PCR. In one study, EGFR amplification was identified in 15.9% of tested cases. Within this group, 71%

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of EGFR FISH-amplified/KRAS wild-type responded to anti-EGFR therapy, versus 37% with normal EGFR copy numbers [45]. Though other studies have found a similar trend, a few studies have opposing findings. Italiano et al. [46] reported that neither EGFR expression assessed by IHC or FISH correlated with an objective response, disease control, PFS, or OS. Additional studies will be required to confirm the clinical utility of these markers; nevertheless, in the near future, we may be seeing “EGFR panels,” which may include some combination of KRAS 12/13/61/146, BRAF, and PIK3CA mutational analysis along with PTEN expression and EGFR gene amplification prior to anti-EGFR therapy.

Conclusions In an era of cost-effective patient management and personalized medicine, KRAS-mutational assays in the management of mCRC serve as a prime example. KRAS codon 12/13 mutational analysis will exclude about 30% to 40% of patients from anti-EGFR therapy, with potential savings in the hundreds of millions of dollars. Unfortunately, a significant proportion of KRAS wild-type tumors do not respond to anti-EGFR therapy, and additional testing of other genes or proteins within the EGFR signaling pathway is likely forthcoming to help guide better patient selection. As the development of management and testing guidelines continues to evolve, “EGFR panels” may become the standard prior to initiating anti-EGFR therapy. Acknowledgment Drs. Patil and Fraser have contributed equally to the manuscript preparation. Disclosure No potential conflicts of interest relevant to this article were reported.

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