Several decades have passed since oncogenic RAS was first identified as the transforming factor in the Harvey and Kirsten strains of the Mouse Sarcoma Virus 1,3-5
. Since these discoveries, all three RAS family genes (KRAS, NRAS
) have been shown to be somatically mutated in human cancer, most commonly as a result of single point mutations at codons 12, 13 and 61.
Despite overwhelming evidence that oncogenic RAS plays a central role in mediating transformation in human tumors, only recently has limited testing for somatic RAS mutations entered routine clinical practice. Widespread adoption of mutational profiling in the clinic has been delayed for several reasons. First, prior to recent advances in sequencing technology, RAS mutational testing was expensive and time-intensive. Second, until recently there was no definitive evidence that routine testing for RAS mutations would meaningfully impact clinical practice. This changed with the identification of KRAS mutations as a predictor of resistance to EGFR kinase inhibitors in patients with lung adenocarcinoma 8
. Similar data soon followed in patients with colorectal cancer, where mutations in exon 2 of KRAS were associated with a lack of clinical benefit with panitumumab and cetuximab 9-13
. On the basis of these data, routine testing of patients with lung and colorectal cancers has become increasingly common, and some clinical practice guidelines and regulatory agencies have proposed the restriction of anti-EGFR therapy to patients whose tumors lack G12 or G13 KRAS mutations.
In the vast majority of studies to date, tumors have been genotyped only for KRAS mutations at the most commonly altered G12 and G13 positions 31-34
. The frequency, predictive, and prognostic value of other RAS mutations has therefore remained poorly defined 16-17
. To facilitate the identification of low frequency RAS pathway mutations, we developed a multiplexed MALDI-TOF based genotyping assay using the Sequenom platform. Consistent with prior studies we observed that approximately one third of colorectal tumors harbored mutations at the G12 and G13 codons. Notably, an additional 10%, that would have been characterized as KRAS wild-type in clinical practice, harbored mutations in exons 3 or 4 of KRAS or in NRAS. These latter mutations were mutually exclusive with those at G12 and G13 suggesting overlapping roles in tumorigenesis.
Our dataset suggests that the underrepresentation of these mutations in the literature and their low reported frequency in the COSMIC database (0.002% of the KRAS mutations reported in the large intestine) is the result of detection bias 35
. To explore this possibility further, we used our MALDI-TOF assay to characterize the frequency of exon 4 KRAS mutations in several additional lineages. In an analysis of 698 non-colorectal cancer tumors and cell lines, we identified only two additional samples with A146 mutations (one ovarian and one endometrial cancer). A146 mutations in KRAS were also not identified in two recent analyses comprising 449 non-small cell lung cancers in which the entire coding region of the gene was sequenced 36-37
. The basis for the higher relative frequency of exon 4 KRAS mutations in colorectal cancer versus other cancers such as lung cancer is unknown, but may be the result of differences in the underlying mutagenic insults responsible for cancer initiation at these sites. We also sequenced exon 4 of both NRAS and HRAS, but detected no mutations in these exons in our colorectal tumors and cell lines. NRAS A146T mutation has, however, been reported in the leukemic cell lines NALM6 and ML-2 16
and germline HRAS mutations at the K117 and A146 codons have been reported in a small number of patients with Costello’s Syndrome 38
The RAS family proteins function as small GTPases that cycle between an inactive GDP-bound and an active GTP-bound state. The slow intrinsic GTPase activity of RAS is enhanced by several orders of magnitude by GTPase activating proteins (RAS GAPs), which include p120 GAP and NF1, which facilitate GTP hydrolysis by stabilizing an intermediate high-energy transitional state 7
. The most common site of RAS mutation located at position 12 results in substitution of glycine for a residue with a side chain. Crystal structure modeling predicts that this substitution is associated with steric interference with GAP-mediated GTP hydrolysis. As glycine is the only amino acid lacking a side chain, a diversity of mutations at this position confer similar phenotypic effects. Mutations at the codon 61 position also impair RAS GTPase activity, but in this case by disrupting a hydrogen bond between the glutamine residue at position 61 of RAS and Arg789 of GAPp120 39-40
Although the mutual exclusivity of the exons 2, 3 and 4 mutations in KRAS suggest significant functional overlap, the cohort of patients with non-exon 2 mutations in KRAS exhibited a better prognosis than patients whose tumors expressed G12 or G13 KRAS mutations. The most common site of KRAS mutation in exon 4 in our series was at amino acid A146. This site is within an evolutionarily conserved region which in structural modeling is predicted to interact with the guanine base of GDP 41
. In contrast to mutations at codons 12 and 13, mutations at codon 146 do not impair RAS GTPase activity 42
. Rather, the transforming potential of the A146 HRAS mutations has been attributed to an increase in guanine nucleotide exchange 42
. As discussed above, mutations of A146 and of the biologically conserved K117 positions of HRAS have been reported in Costello’s syndrome. The most common mutant allele in Costello’s syndrome is G12S and notably, its transforming effects are lower than that of the G12V mutation, which is among the most common mutant alleles in human cancer 43
. Based upon this observation it has been speculated that only low activation alleles of RAS may be compatible with viability when found in the germline.
Given the favorable prognosis of colorectal cancer patients with exon 4 KRAS mutations and the observation that the K117 and A146 mutations are found in Costello’s syndrome, we hypothesized that these mutations may be less potent than mutations at codons 12 and 13. Although we found lower RAS-GTP expression in an isogenic model of K117 and A146T KRAS, in some A146T KRAS expressing cancer cell lines, we observed levels of RAS-GTP comparable to that of cell lines harboring the G12D/V mutations. Our data suggest that whereas A146T KRAS mutation may confer lower intrinsic RAS activity, this may be augmented in part by frequent conversion to homozygosity and low-level copy number gain of the KRAS gene locus.
Our analysis suggests that a broader assessment of RAS mutations beyond the most common mutations in exon 2 is warranted and would lead to the identification of a mutation predicted to confer EGFR inhibitor resistance in close to 50% of patients with colorectal cancer 12,19
. While such testing would decrease the use of toxic and expensive agents in this population unlikely to derive benefit, it would also further limit the available treatment options in a disease in which few currently exist. This and the inability to date to identify a clinically effective inhibitor of RAS have contributed to the reluctance of many clinicians to advocate broader RAS mutation testing. It should be noted, however, that the prospective identification of RAS mutations could also have the secondary benefit of accelerating the clinical development of novel therapies in this class of patients by facilitating the identification of those most likely to benefit. Promising therapeutic approaches include targets that function as synthetic lethals in RAS mutant tumors and inhibitors of downstream effectors such as MEK 30,44-47
. Our data demonstrating complete growth inhibition in A146T KRAS expressing xenografts with the selective MEK inhibitor PD0325901 support the clinical feasibility of this latter approach.
In summary, our data support a more comprehensive assessment of RAS mutational status beyond the most frequently mutated alleles at positions 12, 13 and 61. The ability to use a multiplexed platform makes such an approach feasible even in the case of low frequency alleles. Our data also support the hypothesis that different RAS alleles have overlapping but not identical biologic activities, and may thus confer differential prognostic effects. These differences may impact the choice of therapy in individual cases and may be exploited to therapeutic advantage.