December 2012, Vol 1, No 6
KRAS and Colorectal Cancer: Shades of GrayColorectal Cancer
- Although RAS mutations at glycine-12 and glycine-13 are adjacent, identical substitutions at these positions (eg, G12S vs G13S) lead to very different levels of RAS activation
- The central clinical question remains unanswered: will a patient with metastatic colorectal cancer harboring a KRAS G13D mutation benefit from anti-EGFR therapy?
- If mutations in adjacent codons can have unequal effects, what level of “personalization” is required in oncology clinical research?
Retrospective subset analyses of metastatic or advanced colorectal cancer trials have not shown a treatment benefit for Erbitux or Vectibix in patients whose tumors had KRAS mutations in codon 12 or 13. Use of Erbitux or Vectibix is not recommended for treatment of colorectal cancer with these mutations.
–FDA update to indications and usage of Erbitux (cetuximab) and Vectibix (panitumumab), July 2009.
The association between Kirsten-Ras (KRAS) mutations, present in approximately 40% of colorectal cancers (CRCs), and lack of benefit from epidermal growth factor receptor (EGFR)-targeted antibodies is well known to oncologists. It is standard practice to test for KRAS codon 12 and 13 mutations in tumors of all patients with metastatic CRC (mCRC) because the results affect treatment decisions. Recently, however, 2 reports suggest that KRAS codon 12 and 13 mutations are nonequivalent, although they code for adjacent amino acids.1,2 In cetuximab-treated patients, the presence of a glycine (G) to aspartic acid (D) change at codon 13 of KRAS (c.38G>A, or p.G13D) was associated with intermediate survival, between that of patients with KRAS wild-type tumors and tumors harboring a KRAS mutation at glycine-12. While the overall impact of KRAS mutations on CRC prognosis remains unclear,3 G13D mutations may be associated with worse outcomes.1,2,4
The purpose of this review is to place differences between KRAS mutations at codon 12 and 13 in CRC into scientific and clinical contexts, and to consider the potential implications of nonequivalent mutations within the same gene for the pursuit of personalized medicine in oncology.
The RAS guanine nucleotide-binding protein regulates cell survival, growth, and proliferation by acting as a molecular switch in response to extracellular signals. RAS regulates downstream pathways by cycling between an inactive guanosine diphosphate (GDP)-bound state and an active guanosine triphosphate (GTP)-bound state.5,6 Posttranslational processing targets RAS to the membrane, where it resides in the OFF state, bound to GDP.7,8 Upon stimulation by growth factors such as insulin and EGF, RAS activators called guanine nucleotide exchange factors localize to the membrane and stimulate the release of GDP from RAS, thus allowing GTP binding and activation.9,10 Once ON, RAS activates downstream signaling cascades, including the RAF-MEK-ERK pathway.11
Inactivation of RAS signaling requires hydrolysis of the RAS-bound GTP back to GDP with release of phosphate. However, RAS is extremely slow at carrying out this reaction on its own. Therefore, inactivation of RAS depends upon the action of GTPase-activating proteins (GAPs), which supply a critical arginine residue to the RAS active site to assist in catalysis of GTP hydrolysis.5,12
Dysregulation of RAS signaling is almost invariably associated with disease. Hyperactivating somatic mutations in RAS are among the most common lesions found in human malignancies. Although mutation of any of the 3 RAS isoforms (KRAS, NRAS, or HRAS) can lead to oncogenic transformation, KRAS mutations are by far the most common.13-16 Hyperactivation of the RAS pathway due to oncogenic mutations in other pathway members, including BRAF and EGFR, accounts for a significant proportion of RAS wild-type cancers.17
Three-dimensional structures of RAS isoforms have been well studied. Indeed, over 100 crystal structures of RAS in various complexes have been solved. Nucleotide-dependent signaling relies on conformational changes in 2 regions that border the nucleotide-binding pocket, switch I (residues 30-38) and switch II (residues 60-76) (Figure 1).18 Threonine-35 and glycine-60 make key hydrogen bonds with the ?-phosphate of GTP, holding switch I and switch II in their active conformations. Upon hydrolysis of GTP and release of phosphate, these regions relax into the inactive GDP-binding conformation. Only the GTP-bound conformation of the switch regions activates downstream effector proteins.
The regions surrounding the nucleotide-binding pocket contain the most common sites of RAS mutation. Along with the switch regions, the borders of the pocket include the phosphate-binding loop (P-loop, residues 10-17) and the base-binding loops (residues 116-120 and 145-147).19 The majority of oncogenic mutations occur at residues 12 or 13 in the P-loop, or residue 61 in switch II.17 Mutations activate RAS by 2 main mechanisms: decreasing GTP hydrolysis or increasing nucleotide exchange. Most oncogenic mutations follow the first mechanism. In wild-type RAS, residues 12 and 13 are both glycine, the smallest amino acid. Mutation of either of these residues to larger amino acids can occlude the critical arginine of the GAP, leading to decreased GAP binding and insensitivity to GAP-stimulated GTP hydrolysis.5,20,21 Glutamine-61 of RAS participates in catalysis, and therefore its mutation also affects GTP hydrolysis; however, GAP binding is generally normal or increased.12,21 Mutations near the base-binding region of RAS (eg, alanine-146 or phenylalanine-156) diminish nucleotide binding affinity and increase the intrinsic rate of exchange.22,23 The active form of RAS then predominates due to the higher concentration of GTP than GDP in the cell.
Biochemical and cellular assays demonstrate that although RAS mutations at glycine-12 and glycine-13 are adjacent, identical substitutions at these positions (eg, G12S vs G13S) lead to very different levels of RAS activation.21,24 Mutations at codon 12 are generally the most potent activators of RAS: the crystal structure of HRAS complexed with a GAP suggests that mutations at this position sterically clash with the arginine finger of the GAP, preventing both binding and catalysis.12 Indeed, mutation to any amino acid other than proline induces focus formation and anchorage-independent growth of rat fibroblasts, both measures of oncogenic transformation.24
Mutations at codon 13 are generally less activating in vitro. Three G13 mutations (G13S, G13V, and G13D) have been compared in GAP stimulation and focus formation assays. G13D completely blocks GAP activity and is the only codon 13 mutant that induces focus formation. G13S is only slightly activating, having a small effect on GAP stimulation. G13V shows intermediate oncogenicity; this mutation completely blocks GAP activity, without inducing focus formation.20,21 G12 mutations may confer greater resistance to apoptosis and increase the proportion of GTP-bound RAS well above the level achieved by G13 mutations.1,25-27
Glutamine-61 mutations decrease RAS GTPase activity but vary widely in transformation efficiency.28 Unlike G12 and G13 mutants, some Q61 mutants unexpectedly increase binding to GAP, which could hyperactivate wild-type RAS.21 The most potent mutant, Q61L, increases nucleotide exchange in addition to diminishing GTPase activity.22,29 Substitutions at alanine-146 are less activating than G12 mutations and may be comparable to G13 mutations in transforming efficiency.30 These base-binding mutations increase the intrinsic rate of nucleotide exchange,22 thus increasing RAS-GTP without affecting GAP stimulation. Despite lower oncogenicity, one would expect KRAS A146 mutants to be growth factor independent due to their capacity for autonomous nucleotide exchange and activation.31
The spectrum of RAS pathway mutations differs markedly by cancer type (Figure 2).17 Pancreatic cancer exhibits the highest frequency of KRAS mutations: 72% in the Sanger Catalogue of Somatic Mutations in Cancer and >90% in other series (Figure 2 B).17 By contrast, breast cancer (not shown) has a low frequency of RAS pathway mutations: 4% KRAS, 3% BRAF, 2% NRAS, and <1% HRAS mutations.17 Melanoma has a high frequency of RAS pathway mutations, but KRAS mutations are rare (Figure 2 D). In CRC, half of tumors exhibit a mutation in the RAS pathway (mutually exclusive), most often a missense mutation in KRAS itself (Figure 2 A). Unlike in lung cancer, EGFR mutations are rare in CRC.
Differences by cancer type are also observed in the distribution of KRAS single-base substitution mutations (Figure 2). Across cancers, mutations at codon 12 predominate: G12D and G12V are the first and second most common KRAS mutations. In lung cancer, however, the most frequent KRAS substitution is glycine to cysteine (G12C). Of all cancer types, CRC exhibits the greatest proportion of G13D mutations: ~20% of KRAS mutations and 8% overall. Of note, clinical testing for KRAS mutations is typically performed using polymerase chain reaction–based assays with probes specific for mutations in codons 12 and 13. Consequently, KRAS mutations at other codons, including Q61 and A146, may be underrepresented.32,33
Despite decades of RAS research, 5 years passed between FDA approval of cetuximab for treatment of EGFR-expressing mCRC and the label change to exclude patients with tumors harboring KRAS mutations. A major reason for the delay was that no correlation was observed between KRAS mutational status and sensitivity to cetuximab in CRC cell lines.34 This experience underscores the necessity of evaluating annotated human tumor specimens for associations between specific mutations, survival, and response to therapies, thereby enabling the clinical findings to guide further mechanistic investigations.
A landmark study linking KRAS G13D mutations with clinical outcomes was a retrospective, pooled analysis of 774 patients with chemotherapy-refractory mCRC treated with cetuximab-based therapy from 3 data sets (the National Cancer Institute of Canada/Australasian Gastrointestinal Trials Group and a Leuven and an Italian data set) encompassing 7 clinical trials (CO.17, EVEREST, BOND, SAVAGE, BABEL, MABEL, and EMER202600).1
Three hundred ten patients (40%) had a KRAS mutation at codon 12 or 13, 45 of which were G13D (14.5% of KRAS mutations; 6% overall). Thirteen patients with KRAS G13D-mutated tumors received best supportive care on the CO.17 trial and experienced significantly worse overall survival (OS) compared with patients with other KRAS-mutated or KRAS wild-type tumors in a univariate analysis. After adjustment for potential prognostic factors (eg, age, sex, performance status, prior chemotherapy, and primary site) in the multivariate analysis, however, there were no survival differences by KRAS status (Table). Patients with G13D-mutated tumors who received any cetuximab-based therapy (monotherapy or cetuximab plus chemotherapy) experienced longer survival than patients with other KRAS mutations, with OS 7.6 versus 5.7 months and progression-free survival (PFS) 4.0 versus 1.9 months (Cox regression P=.005 and .004, respectively) (Table). But because G13D did not portend a worse prognosis in multivariate analysis, the significance of improved survival end points for cetuximab-treated patients with G13D-mutated tumors compared with other KRAS mutations is unclear. Moreover, all of the benefit occurred in patients treated with cetuximab plus chemotherapy, raising the possibility that outcomes could have been more affected by the chemotherapy. In the presence of a G13D mutation, cetuximab monotherapy (pooled data) did predict significantly better OS compared with no cetuximab (CO.17 trial) (P=.02 by log-rank test), and there was a trend toward significance in the CO.17 trial only. There was no significant difference in response rates between G13D and other KRAS mutants treated with cetuximab-based therapy. Overall, the results of De Roock et al1 provide greater support for G13D as a predictive rather than a prognostic marker.
Following these findings in the refractory setting, the association of KRAS G13D with outcomes in patients with mCRC treated with first-line chemotherapy with or without cetuximab was investigated by Tejpar et al in a retrospective analysis of 1378 patients from the randomized CRYSTAL and OPUS trials.2 Here, 533 patients (39%) had a KRAS mutation at codon 12 or 13, 83 of which were G13D (16% of KRAS mutations; 8% overall). Median OS was longer than in De Roock et al,1 consistent with first-line therapy, compared with the chemotherapy-refractory setting (Table). When treated with chemotherapy alone, patients with G13D mutant tumors had a worse outcome compared with patients with other KRAS mutant or KRAS wild-type tumors. When cetuximab was added to chemotherapy, significant improvements in PFS (median 7.4 vs 6.0 months, hazard ratio [HR] 0.47; P=.039) and tumor response (40.5% vs 22.0%; odds ratio 3.38; P=.042) were observed among patients with G13D mutations, although the OS benefit did not reach significance (Table). The cetuximab treatment effect in the G13D subset was comparable to that in patients with KRAS wild-type tumors, but OS and PFS were considerably lower. By contrast, survival worsened with the addition of cetuximab to chemotherapy among patients with other KRAS mutations (6.7 vs 8.1 months; HR 1.37).
To summarize, both of these large, pooled retrospective studies found that patients with KRAS G13D mutant mCRC may have a poor prognosis when treated with best supportive care or standard chemotherapy in the chemotherapy-refractory1 or first-line setting,2 respectively. In both studies, patients with G13D mutations appeared to derive modest benefit from the addition of cetuximab.
Smaller, single-arm studies have yielded complex results with respect to the prognostic significance of KRAS G13D mutations, a confounding factor that clouds interpretation of predictive impact.4,35 Evaluation of 229 Japanese CRC patients found a nonsignificant trend toward worse OS of patients with G13D mutations compared with patients with KRAS wild-type tumors (HR 1.67; 95% CI, 0.93-3.02; P=.086 on univariate analysis).4 Survival of patients with G12 mutant tumors was similar to KRAS wild-type, whereas patients with BRAF V600E mutant tumors had the worst OS (HR 3.78; 95% CI, 1.89-7.54; P?.001).4 On the other hand, 2 reports suggest a link between KRAS G13D and microsatellite instability (MSI) associated with favorable outcome.35,36 An analysis of mutation frequency in 158 hereditary nonpolyposis CRC (HNPCC) and 864 sporadic CRC cases identified a significantly higher rate of G13D mutations in HNPCC tumors (microsatellite unstable) compared with sporadic microsatellite-stable tumors (51% vs 12% of KRAS mutations).36 G13D mutations were also overrepresented in sporadic MSI-high tumors without hMLH1 methylation (27% of KRAS mutations).36 In a study of 94 hereditary and 404 sporadic CRC cases, KRAS G13D mutations were associated with a better prognosis in HNPCC and sporadic MSI-high subsets.35
A growing number of small studies are examining the predictive value of KRAS G13D with respect to cetuximab treatment.37-39 These studies, limited by size and clinical heterogeneity, highlight the importance of access to tissue from large groups of similarly treated patients for subset analyses.
Peeters et al40 presented an important counterpoint to the findings in De Roock et al1 and Tejpar et al2 at the 2012 American Society of Clinical Oncology (ASCO) Annual Meeting. Evaluation of 1083 patients treated with second-line FOLFIRI plus panitumumab (NCT00339183), 1096 patients treated with first-line FOLFOX4 plus panitumumab (PRIME, NCT00364013), and 184 patients who received panitumumab monotherapy (NCT00113776) yielded inconsistent associations between specific KRAS mutations and survival.40 Tejpar et al2 pooled data from the CRYSTAL (FOLFIRI ± cetuximab) and OPUS (FOLFOX4 ± cetuximab) studies because they found that irinotecan- and oxaliplatin-containing chemotherapy regimens produced similar treatment effects in patients stratified by KRAS mutation status. Conversely, Peeters et al40 reported a trend toward better OS among patients with G13D-mutated tumors treated with FOLFIRI plus panitumumab (N=39; P=.07), whereas G13D was significantly associated with worse OS among patients treated with FOLFOX4 plus panitumumab (N=46; P=.003), and G13D was not predictive or prognostic of outcome when the panitumumab studies were pooled. The possibility that different anti-EGFR agents, in addition to lines of therapy and chemotherapy backbones, may contribute to unequal outcomes in association with KRAS mutation status further complicates the G13D story.
More Questions Than Answers
Following these analyses of over 3000 patients, evidence to support KRAS G13D as an independent predictive and/or prognostic biomarker in CRC still has not matured. Returning to the question of biologic mechanism, is there a plausible explanation for intermediate effects of KRAS G13D with respect to both response to cetuximab and survival? Intermediate response to cetuximab is easier to explain because KRAS mutations at codon 13 are less oncogenic than mutations at codon 12.1,20,21,24 A less activating KRAS mutation may mean that the tumor is partially growth factor dependent and thereby somewhat responsive to EGFR inhibition. It is difficult to directly attribute worse survival to G13D mutations. More likely, G13D mutations could signify a different biological signature that portends worse prognosis.
Different carcinogenic triggers produce distinct patterns of mutations: depending on the chemical used, KRAS codon 12 or 61 mutant lung cancers can be induced in mice.41 In human lung cancers, KRAS mutations are common in smokers, whereas EGFR and ALK mutations are more frequent in never-smokers.42 In CRC, rather than being a driver, KRAS G13D mutations may develop secondary to another oncologic process and propagate because they confer a modest survival advantage for tumor cells. This hypothesis is supported by findings in cultured cells subjected to continuous cetuximab: KRAS G13D mutations accompanied emergence of resistance, and deep sequencing subsequently detected preexisting G13D mutations in a minority of parental cells.43 KRAS G13D is also a nontumor-initiating mutation in HNPCC, caused by defects in DNA mismatch repair genes that lead to a myriad of somatic mutations.36,44 A poor prognosis, BRAF-mutated–like gene expression signature was identified in 30% of KRAS mutant CRCs.45 Follow-up work presented at the 2012 ASCO Annual Meeting showed additional evidence for heterogeneity among KRAS mutant colon tumors but no significant division by specific KRAS mutation.46 A moderately aggressive CRC subgroup marked by KRAS G13D mutations has not been described.
If “shades of gray” exist among KRAS mutations, might the same be true for other oncogenes and tumor suppressor genes? The answer is, of course, yes. For example, phosphoinositide-3-kinase (PI3K, coded by the PIK3CA gene) is mutated in ~15% of CRCs. PIK3CA has 2 mutational hot-spot clusters located approximately 1500 nucleotides apart, corresponding to the helical domain (eg, E545K) and the kinase domain (eg, H1047R). Helical and kinase domain mutations induce gain of function by different mechanisms. Helical domain mutations are dependent on RAS binding for transformation; kinase domain activation is mediated by interaction with the p85 regulatory subunit.47
In CRC, helical domain mutations associate with KRAS mutations, methylation of the MGMT DNA repair gene, and CpG island methylator phenotype–low status, while kinase domain mutations correlate with serrated pathway features including BRAF mutation and MSI.48 As with KRAS, the distribution of PIK3CA helical and kinase domain mutations varies by tissue of origin. In CRC, the distribution is roughly two-thirds helical to one-third kinase domain mutations. Breast cancer has the highest rate of PI3K mutations (25%), and the ratio of helical to kinase domain mutations is opposite that in CRC.17 Even identical mutations can have differing effects by cancer type, as highlighted by dissimilar responses to BRAF inhibitors among patients with BRAF V600E CRC or melanoma.49
A final example of the potential for disparate impact of mutant alleles within a single gene involves the tumor suppressor protein p53. Mutation of p53 is the most common genetic lesion in human malignancies, including nearly half of CRCs. Whereas a limited number of mutations activate oncogenes, a greater variety of lesions can inactivate tumor suppressors. One study assessed effects of 2314 point mutations in the p53 DNA-binding domain.50 We ascertained p53 status in 604 tumors from the CALGB 89803 trial of adjuvant therapy for stage III colon cancer. Tumors were classified as p53 wild-type or mutation present in the zinc-binding (ZB) or the non–zinc-binding (NZB) regions of the DNA-binding domain. Overall, p53 mutational status was not associated with survival.51 However, among women, survival was superior with NZB mutations compared with wild-type p53 and worst with ZB mutations.51 A larger sample would be required to determine whether these differences could be attributed to specific p53 hot-spot mutations.
Implications for Personalized Medicine
The central clinical question remains unanswered: will a patient with mCRC harboring a KRAS G13D mutation benefit from anti-EGFR therapy? Based on the modest efficacy of anti-EGFR therapy even among patients with KRAS wild-type tumors, the sample size required for a prospective randomized study in the KRAS G13D subset would be prohibitive and perhaps inappropriate in light of the priority to identify new targets and more active agents. Additional large retrospective analyses of cohorts of CRC patients with matched clinicopathologic and treatment characteristics may provide greater precision in estimating the prognostic and predictive value of G13D, although there remains a distinct possibility that the impact is unsatisfyingly context-dependent and marginal. Are the differences meaningful, or are we trying to distinguish gray from grey?
KRAS G13D heralds an approaching onslaught of new questions as next-generation sequencing and other technologies reach the clinical arena. If mutations in adjacent codons can have unequal effects, what level of “personalization” is required in oncology clinical research? Even synonymous mutations (resulting in the same amino acid sequence) can produce differences in RNA processing that significantly alter protein levels or functions.52 We submit that validation of novel “personalized medicine” biomarkers should involve an iterative approach whereby hypothesis-generating data from clinical samples – such as the observations reviewed here suggesting a differential impact of KRAS G13D versus other KRAS mutations – should trigger a return to preclinical mechanistic studies in parallel with additional retrospective analyses of independent clinical data sets. Across these endeavors, tumor genotyping should be specific to the nucleotide level and compiled to identify broad signals of high-impact genetic events. As our understanding of CRC biology becomes more nuanced, we must steadfastly search for therapies with unequivocal benefit for molecularly defined subsets of patients.
CEA is supported in part by Postdoctoral Fellowship 11-183-01-TBG from the American Cancer Society and also acknowledges the support Millennium provided through the Alliance for Clinical Trials in Oncology Foundation. JMO is supported by the UCSF Medical Scientist Training Program NIH-NIGMS-MSTP (#GMO7618). We thank David Donner, PhD, for editorial assistance.
- De Roock W, Jonker DJ, Di Nicolantonio F, et al. Association of KRAS p.G13D mutation with outcome in patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab. JAMA. 2010; 304:1812-1820.
- Tejpar S, Celik I, Schlichting M, et al. Association of KRAS G13D tumor mutations with outcomes in patients with metastatic colorectal cancer treated with first-line chemotherapy with or without cetuximab [published online ahead of print June 25, 2012]. J Clin Oncology.
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