Abstract: The impact of KRAS mutations on the efficacy of therapies that target the epidermal growth factor receptor (EGFR) is a major, ongoing area of oncology research, aimed at identifying the best possible treatments for individual colon cancer patients. Because patients with KRAS mutant colorectal tumors rarely respond to anti-EGFR monoclonal antibodies, testing is required to confirm the patient's tumor is KRAS wild-type before utilizing these therapies. Despite being studied for more than 30 years, new information continues to develop regarding KRAS and its role in colon carcinogenesis. This information must be integrated into the development of effective colon cancer treatment strategies. This review will summarize recent evidence that most, if not all, colon tumors encompass at least a subpopulation of KRAS mutant cells, meaning tumors characterized as KRAS wild-type are in most cases tumors with relatively low KRAS mutant tumor cell content. Recent studies support the hypothesis that relapse in advanced colorectal patients treated with EGFR-targeted monoclonal antibody therapy involves the outgrowth of previously undetected KRAS mutant tumor cell populations. Studies investigating the effects of oxidative stress on Ras signaling suggest that the frequent presence of minor KRAS mutant tumor cell populations may be a consequence of hypoxic conditions within tumors, which produce a negative selection against KRAS mutant cells in polyclonal tumors. Thus, the literature and current practices for characterizing tumor KRAS mutation don't accurately reflect the nature of colon tumor KRAS mutation, even though an accurate understanding is critical for identifying the best strategies for intervention.
The objective of personalized cancer treatment is to select the ideal therapy for an individual cancer patient based on knowledge of each patient’s tumor characteristics and/or genetics. High expectations for improving cancer outcomes through personalized cancer treatment arose from patient responses to the cancer drug imatinib, which is a tyrosine-kinase inhibitor. The survival of patients with chronic myelogenous leukemia, who carry a particular translocation in their leukemic white blood cells, was extended significantly by treatment with imatinib (Horne et al., 2013). More recent experience, applying the concept of personalized cancer treatment to solid tumors, has been less encouraging. In particular, a great deal of clinical research has been focused on drugs and biologics that target the epidermal growth factor receptor (EGFR). In the treatment of advanced colorectal cancer, it was discovered that patients whose tumors carry a KRAS mutation are much less likely to respond to antibodies that block EGFR signaling (Baldus et al., 2010; Bando et al., 2011; De Roock et al., 2010a; Freeman et al., 2009). These findings led to a requirement for KRAS mutational testing of metastatic colorectal cancer (mCRC) and restricting the use of the anti-EGFR monoclonal antibodies cetuximab and panitumumab to patients with KRAS wild-type mCRCs (Heinemann et al., 2009; Linardou et al., 2011).
On its face this represents another advance toward effective personalized cancer treatment (Heinemann et al., 2013). Yet, the increases in progression-free survival (PFS) for patients with KRAS wild-type tumors are modest (Amado et al., 2008; Bando et al., 2011; Molinari et al., 2011), half of patients with KRAS wild-type tumors fail to respond (Pentheroudakis et al., 2013), and more importantly, eventually all patients develop resistance to treatment and relapse (Misale et al., 2012). This review will summarize the evidence from which: 1) it can be concluded that most, if not all, colon tumors carry abnormal levels of KRAS mutation, meaning few if any colon tumors are actually KRAS wild-type, and 2) the use of EGFR-targeted therapies to treat predominantly KRAS wild-type tumors creates an opportunity for the outgrowth of KRAS mutant subpopulations, which can lead to acquired resistance to treatment and relapse. The significance of these findings will be discussed in terms of their broader implications for developing effective personalized cancer treatments.
Mutational Analysis: Lessons from the Field of Genetic Toxicology
Mutational analysis is an area of endeavor in which many oncologists may have little practical experience. Consequently, it is worthwhile to consider some general knowledge gleaned from decades of study in the field of genetic toxicology. Genetic toxicology encompasses a broad array of techniques developed to determine whether or not a particular chemical treatment or exposure causes genetic damage. Because such methods are used to measure genetic consequences of short-term chemical exposures, including somatic mutations, the methods need to be quite sensitive. Some mutation detection methods, listed in order of increasing sensitivity, are: DNA sequencing, single-strand conformation polymorphism analysis, denaturing gradient gel electrophoresis, and restriction fragment length polymorphism PCR. Also, there are a wide variety of allele-specific PCR methods, which vary in sensitivity from those that can detect one mutant per 40 wild-type molecules to those that can detect one mutant per 105 wild-type molecules (McKinzie and Parsons, 2002; Parsons and Heflich, 1997; Parsons et al., 2010a). Those in the field of genetic toxicology realize that: 1) the more sensitive the mutation detection method employed, the more mutations will be identified, and 2) that with sufficient sensitivity, it is possible to measure the spontaneous mutations present in DNA isolated from normal, untreated tissues (Parsons and Heflich, 1997; Parsons et al., 2010b). These facts also apply to the detection of tumor mutations in clinical samples, as is apparent from the literature (Molinari et al., 2011).
The Frequency of Tumor KRAS Mutations Reported in the COSMIC Database vs. Data Collected Using Sensitive Mutation Detection Methods
In the context of personalized cancer treatment, tumor mutational analysis by DNA sequencing has been described frequently as the “gold standard” (Baskin et al., 2011; Blank et al., 2011; Davidson et al., 2012; Heinemann et al., 2009; Linardou et al., 2011), yet DNA sequencing is one of the least sensitive methods for characterizing mutation. At least 10-20% of a DNA population must be mutant for the mutation to be detected by standard DNA sequencing (Carotenuto et al., 2010; Ogino et al., 2005). Implicit acceptance that this level of sensitivity is sufficient to characterize tumor mutations only makes sense if one assumes that tumors are monoclonal and that all the critical driver mutations will be present in the bulk of the tumor. Yet these are incorrect assumptions. In fact, it has become clear that most if not all colon tumors are polyclonal and heterogeneous (Parsons, 2008; Thirlwell et al., 2010; Zeki et al., 2011). Consequently, tumor driver mutations may not be detectable using standard DNA sequencing methods.
Currently available evidence proves that many colon tumors, which would be characterized as KRAS wild-type by DNA sequencing, actually contain KRAS mutant subpopulations. Numerous studies have characterized the KRAS mutational status of colon tumors by DNA sequencing. Generally, such studies report that ~30% of colon tumors are KRAS mutant (Prior et al., 2012). This is borne out by the data in the COSMIC database (Catalogue of Somatic Mutations in Cancer; www.sanger.ac.uk/ genetics/CGP/cosmic/). A survey of the most recently released COSMIC dataset shows that 37% of colonic adenocarcinomas carry KRAS mutation and 63% are KRAS wild-type (See Figure 1A). Although not apparent from Figure 1A, 52/6894 colon tumors in the COSMIC database were found to carry more than one KRAS mutation. Using a more sensitive mutation detection method (polymerase chair reaction-restriction fragment length polymorphism) it was shown that 11/74 (15%) of colon tumors had KRAS codon 12 or 13 mutant:wild-type ratios of >0.25, but another 58/74 (78%) of colon tumors had KRAS mutant:wild-type ratios between 10-3 and 10-1 (Dieterle et al., 2004). From the data in this study, therefore, it can be concluded that 93% of colon tumors possessed at least one KRAS mutant cell per 500 wild-type cells (assuming one mutant copy per cell, a mutant:wild-type ratio of 10-3 corresponds to one mutant per 500 non-mutant cells). Allele-specific competitive blocker PCR (ACB-PCR), with a sensitivity of 10-5, was also used to quantify levels of two frequently occurring KRAS mutations in colon tumors, KRAS codon 12 GAT (G12D) and GTT (G12V) (Parsons et al., 2010a). ACB-PCR is sensitive enough to quantify the spontaneous KRAS mutation present in normal colonic mucosa samples (Parsons et al., 2010a; 2010b), so ACB-PCR detection of KRAS mutation does not necessarily signify a pathological level of mutation. Therefore, the 95% upper confidence interval for the level of KRAS mutation measured in normal colon tissue was calculated (from published and unpublished data) and is provided as a point of reference for the KRAS mutant fractions (ratios of mutant:wild-type DNA) measured in individual tumors (see Figure 2) (Parsons et al., 2010a). It was observed that 11/37 (30%) of colon tumors (adenomas and adenocarcinomas) carried a KRAS codon 12 GAT or GTT mutation at a level of 10‑1 or above (i.e., that detectable by DNA sequencing). Importantly, 26/37 (70%) of the colon tumors carried at least one of the two mutations measured at a level above the upper 95% confidence interval for that present in normal colonic mucosa (see Figure 2). In addition, it was determined that 30% of colon tumors carried abnormal levels of both mutations (see Figure 1B). Only two of the most prevalent KRAS mutations were analyzed by ACB-PCR. KRAS codon 12 GAT (G12D) and GTT (G12V) account for 57% of the KRAS mutant colonic adenocarcinomas in the COSMIC database (see Figure 1A). Because 70% of colon tumors carry at least one of these two mutations as a subpopulation (see Figure 1B), and these two mutations account for only 57% of the known KRAS mutations (see Figure 1A), it can be hypothesized that if all possible KRAS mutations were measured with the sensitivity of ACB-PCR, then most, if not all, colon tumors would be found to contain at least subpopulations of KRAS mutant cells. This hypothesis is supported by the previously discussed report indicating a sensitivity of 10-3 detects KRAS mutation in 93% of colorectal cancers (Dieterle et al., 2004). Thus, KRAS wild-type tumors are rare or non-existent (rather than the frequently cited values of 60-70%), and it is not uncommon for colon tumors to carry more than one KRAS mutation.
Additional evidence that colon tumors contain KRAS mutant subpopulations is derived from studies that uncovered intratumoral heterogeneity in KRAS mutation by analyzing multiple, spatially distinct specimens of individual tumors (Baldus et al., 2010; Mancuso et al., 2010; Richman et al., 2011).
Evidence KRAS Mutant Subpopulations Affect Patient Outcomes
Because it has been established that undetected KRAS mutant tumor subpopulations are frequently present in colon tumors, the critical question becomes whether low frequency mutations have clinical impact. The results of several studies indicate KRAS mutant subpopulations are impacting clinical responses to therapies that target EGFR. Two studies employed KRAS mutational testing of colorectal tumors by multiple methods and reported that when KRAS was characterized using a more sensitive method than DNA sequencing better concordance between KRAS mutational status and patient outcome was observed (Bando et al., 2011; Molinari et al., 2011). This shows that mutant KRAS levels below those detectable by DNA sequencing can impact patient responses.
Additional evidence that KRAS mutation is a driver of acquired resistance to therapies that target EGFR is derived from studies that followed patients being treated with anti-EGFR monoclonal antibodies and report expansion of previously undetected KRAS mutants. Misale et al. (2012) confirmed that KRAS mutation confers resistance to cetuximab in vitro. They used the high-sensitivity BEAMing technique to show 7/11 patients who developed resistance to cetuximab or panitumumab had previously undetected KRAS mutant tumor subpopulations and mutant KRAS DNA in their sera. Similarly, the appearance of KRAS mutation in the sera of patients five to six months following initiation of panitumumab treatment was observed in patients previously found to have KRAS wild-type tumors (Diaz et al., 2012). The conclusion from these studies is that emergence of KRAS mutant subpopulations is a mediator of acquired resistance to EGFR blockade (Misale et al., 2012). There are additional mechanisms by which colon tumors can acquire resistance to EGFR-targeted therapies (Alexander and Friedl, 2012), including mutations in the downstream molecules of the mitogen-activated protein kinase pathway (De Roock et al., 2010a; Heinemann et al., 2013). Yet, it can be argued that KRAS mutant subpopulations are a primary driver of relapse in patients treated with EGFR-targeted therapies, based on the prevalence of KRAS mutant subpopulations (see Figures 1 and 2), and the minor fraction of patients with KRAS mutant mCRC who respond to EGFR blockade (De Roock et al., 2010a; Freeman et al., 2008).
Different KRAS Mutations Have Different Impact on Patient Response to EGFR-targeted Therapies
The multicenter RASCAL study was the first to correlate specific colorectal tumor mutations with patient prognosis and reported that the KRAS G12V amino acid substitution was an independent risk factor for poor survival (Andreyev et al., 1998). Additional studies have confirmed KRAS codon 12 mutations confer a poor prognosis (Pentheroudakis et al., 2013), but also provide evidence that the specific KRAS mutation determines the nature of the response to EGFR blockade. A number of studies have investigated whether patients with KRAS codon 13 mutant tumors respond better than those with KRAS codon 12 mutations [for a review see (Amatu et al., 2012)]. These encompass a variety of different treatment conditions and patient populations, so it is not surprising the results are mixed, but in patients with chemotherapy-refractory mCRC treated with cetuximab, longer overall survival (OS) was observed for patients with KRAS G13D mutant tumors than for patients whose tumors carried other KRAS mutations (De Roock et al., 2010b). In mCRC patients treated with cetuximab in combination with capecitabine plus oxaliplatin or capecitabine plus irinotecan as first-line treatment, those whose tumors carried KRAS codon 13 mutations had significantly longer PFS and OS than those with KRAS codon 12 mutations (Modest et al., 2012). A pooled analysis of three trials concluded that patients with KRAS G12D tumors showed a trend of favorable outcomes to cetuximab-based first line therapy compared to other mutations, whereas patients with KRAS G12C tumors had unfavorable outcomes when treated with oxaliplatin plus cetuximab.
These results are not surprising given that different amino acid substitutions affect RAS protein structure and its consequent signaling capabilities. It is known that the different KRAS mutations confer different phenotypic properties to RAS. For example, the KRAS GTPase activity of G12V was found to be one-quarter that of G12D and one-tenth that of wild-type KRAS (Al-Mulla et al., 1999). Using stably-transfected cells lines, it was shown that growth rates of KRAS G12V and G12D transfectants were similar, but greater than that of KRAS G12C transfectants, which was greater than that of KRAS wild-type control cells (Cespedes et al., 2006). In addition, KRAS G12V transfectants were more tumorigenic in nude mice than G12D transfectants (Cespedes et al., 2006). The functional differences associated with particular amino acid substitutions also cause differences in signaling pathway activation; specifically, the KRAS G12V protein interacts with Raf-1 and signals through the Erk pathway, whereas KRAS G12D protein does not interact with Raf-1 and instead signals primarily through the PI3K/Akt, JNK, p38, and FAK pathways (Cespedes et al., 2006). Differential downstream RAS signaling by different RAS proteins (wild-type, G12D mutant, and G12C mutant) has been documented in lung cancers (Ihle et al., 2012), but not yet in colorectal cancers. Recently, using x-ray crystallography, the role of an allosteric switch mechanism in RAS protein structure was described as a regulator of signaling through the Ras/Raf/MEK/ERK pathway (Buhrman et al., 2011). Finally, a recent study of cancer cell lines showed that within a cell KRAS wild-type and mutant isoforms have independent and nonredundant roles in regulating signaling networks (Young et al., 2013).
Why Are KRAS Mutant Tumor Subpopulations So Prevalent in Colon Tumors?
It is not known why KRAS mutation is so frequently found in colon tumors as subpopulations, but two clues have been gleaned from the quantification of KRAS mutation within tumor DNA samples. Specifically, KRAS codon 12 GTT mutation levels (G12V) were shown to decrease during adenoma to adenocarcinoma progression (Parsons et al., 2010a), and a significant, inverse association was observed between maximum tumor dimension and KRAS codon 12 GTT mutation level (Parsons and Myers, 2013). These observations, coupled with studies showing associations between hypoxia in tumors, RAS signaling, and the role of reactive oxygen species (ROS) in regulating proliferation and senescence (DeNicola and Tuveson, 2009; Lu and Finkel, 2008; Ralph et al., 2010) suggest that hypoxic conditions in growing tumors generate a negative selective pressure against KRAS mutant clones, thereby explaining their frequent occurrence as mutant subpopulations (Figure 3). Specifically, hypoxia and/or glucose deprivation, which may occur at particular regions of large advanced tumors (Alexander and Friedl, 2012; Kizaka-Kondoh et al., 2009), may activate RAS signaling and lead to the production of ROS (DeNicola and Tuveson, 2009; Lu and Finkel, 2008; Ralph et al., 2010). Cellular signaling is altered by prolonged exposure to ROS and oxidative stress, and may lead to oncogene-induced senescence or even selective killing of KRAS mutant cells (Shaw et al., 2011). Thus, there is a potential mechanism by which the relative abundance of KRAS mutant cells may decrease in large, advanced cancers. In addition, the senescence-associated secretory phenotype may explain how minor KRAS mutant tumor cell populations can drive tumor progression (Coppe et al., 2008). Senescent KRAS mutant cells were shown to be capable of inducing hyperproliferation, epithelial-mesenchymal transition, and invasiveness by secretion of inflammatory cytokines that alter the microenvironment (Coppe et al., 2008).
Future Directions and Challenges
Oncology drug development is one of the most problematic areas of drug development, with less than 25% of the oncology drugs that enter clinical testing ever obtaining marketing approval from the U.S. Food and Drug Administration (DiMasi and Grabowski, 2007). This likely reflects the fact that the pre-clinical models being used to select drug leads and establish efficacy do not accurately capture the complexity of the disease to be treated (Humphrey et al., 2011), specifically, a complex integrated population of interdependent clones carrying a variety of different genetic lesions within a tumor microenvironment that has lost the ability to maintain normal tissue homeostasis. If this view of tumor biology is embraced and combined with new technological advances, the future may bring more rapid development of cancer therapeutics. For example, 3D primary tumor spheroid cultures, a model that captures the complexity of heterogeneous tumors and their extracellular matrix, may be an appropriate model for assessing the efficacy of potential therapeutics (Kondo et al., 2011).
It has long been recognized that there are “driver mutations” and “passenger mutations,” and it has been assumed that critical driver mutations could be detectable using relatively insensitive methods, like DNA sequencing. The example of KRAS shows that there is a subcategory of cancer driver mutations, which could perhaps be designated as “transdriver mutations,” that can drive tumor progression and impact clinical response, even when they are present as only minor subpopulations within tumors. Given the conclusion that most if not all colon tumors encompass KRAS mutant subpopulations, developing combination therapies that block the outgrowth of these subpopulations should be a research priority.
Despite being studied for so many years, research is uncovering new insights into RAS protein structure, the protein regulators of RAS, and the downstream signaling pathways regulated by RAS, along with new concepts regarding the role of KRAS mutation in colon carcinogenesis. It is clear that KRAS mutation is not just one of many cancer driver mutations that may contribute to colorectal cancer if it should occur by chance. An updated view of KRAS in colon carcinogenesis recognizes that there is a relatively high spontaneous background levels of KRAS mutation in normal human colon tissue. KRAS mutant cells are likely held in check by normal homeostatic mechanisms, as proposed by Bissell and Hines (2011). When those fail, perhaps due to the occurrence of co-localized, cooperating genetic, or epigenetic lesions, the pre-existing KRAS mutants in normal tissue may contribute to polyclonal colon tumor initiation, with additional mutations in KRAS and other genes arising during tumor progression. With increasing tumor size, hypoxia may produce a negative selection against KRAS mutant cells, leading to the frequent occurrence of KRAS mutant cells as tumor subpopulations. As subpopulations, these KRAS mutant cells may continue to drive cancer progression through a senescence-associated secretory phenotype.
This updated view of the role of KRAS mutation in colon carcinogenesis is consistent with currently available data and has important implications for the development of effective personalized cancer treatments. It has become clear that monotherapies directed at the non-KRAS mutant bulk of polyclonal tumors leads to the outgrowth of KRAS mutant subclones and eventually to relapse. Given the prevalence of KRAS mutation in colon tumors as detected by DNA sequencing, along with the frequency of tumors that carry KRAS mutant subpopulations, it can be concluded that most, if not all, colon tumors carry KRAS mutations. Therefore, there is a critical need for effective therapeutics that target KRAS mutant cells, either directly or through its downstream effectors. It is envisioned that such therapeutics could be routinely incorporated into the treatment of colon cancer patients, in combination with additional therapeutics that target specific cancer susceptibilities within a given patient’s tumor.
The authors thank Drs. Malathi Banda and Page McKinzie for their critical review of the manuscript. The opinions and information in this review article are those of the authors, and do not represent the views and/or policies of the U.S. FDA, nor does the mention of trade names or commercial products constitute endorsement or recommendation for use.
The authors report no conflicts of interest.
Barbara L. Parsons, Ph.D., Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, HFT-120, 3900 NCTR Rd., Jefferson, Arkansas 72079, USA.
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[Discovery Medicine; ISSN: 1539-6509; Discov Med 15(83):259-267, April 2013. Copyright © Discovery Medicine. All rights reserved.]