Advances in Anatomic Pathology:
KRAS Mutation Testing in Colorectal Cancer
Plesec, Thomas P. MD; Hunt, Jennifer L. MD, MEd
Department of Anatomic Pathology, Cleveland Clinic, Cleveland, OH
Reprints: Thomas P. Plesec, MD, Department of Anatomic Pathology, Cleveland Clinic, L25, 9500 Euclid Avenue, Cleveland, OH 44195 (e-mail: email@example.com).
All figures can be viewed online in color at http://www.anatomicpathology.com
In the US, colorectal cancer is the third leading cause of cancer-related death. Approximately 20% of patients present with metastatic disease, and an additional 30% to 40% develop metastasis during the course of their disease. Patients with metastatic colon cancer have a 5-year survival rate of only 11%. Although surgery is the mainstay of treatment for early stage colon cancer, adjuvant treatment is usually used in patients advanced stage disease. In particular, epidermal growth factor receptor (EGFR) inhibitor therapies have emerged as effective treatments in a subset of patients with metastatic colorectal carcinoma. Two anti-EGFR biologics, cetuximab and panitumumab, have been approved by the Food and Drug Administrations for the treatment of refractory metastatic colorectal carcinoma. Mounting evidence has shown that these therapies are ineffective in tumors with mutations of codons 12 and 13 of exon 2 of the KRAS gene. Because of this compelling data, the National Comprehensive Cancer Network and the American Society of Clinical Oncology have recommended determination of KRAS mutation status in all patients with metastatic colorectal cancer who are candidates for anti-EGFR therapy. Anatomic pathologists play an integral role in coordinating the testing for KRAS mutations, as this assay is performed on tissue samples selected by the pathologist. Herein, the authors present an up-to-date review of the biologic, clinical, and laboratory aspects of KRAS mutation testing in colorectal cancer.
EPIDERMAL GROWTH FACTOR RECEPTOR AND COLORECTAL CANCER
Epidermal growth factor receptor (EGFR), also known as HER1/ErbB1, is a member of the HER family of transmembrane cell surface receptors, is found in most epithelial tissues, and is involved in the regulation of cell growth and cell cycle progression (Fig. 1).1 EGFR responds to ligands such as epidermal growth factor, transforming growth factor-α, and ampiregulin, among others.2 Ligand binding leads to homodimerization with another EGFR or heterodimerization with other HER family receptors. Dimerization initiates autophosphorylation of intracellular tyrosine residues,3 which leads to activation of various signaling pathways, including RAS-mitogen-activated protein (MAPK), 3-kinase (PI3K)-AKT, and Janus-activated kinase-STAT.4
EGFR is integral to the growth and survival of many solid cancers, including colorectal cancer. EGFR, as measured by immunohistochemistry, is overexpressed in up to 82% of colorectal cancers,5,6 and both EGFR and epidermal growth factor levels are markedly higher in carcinoma than in adjacent non-neoplastic mucosa.7 Enhanced ligand response or complete ligand independence result in over activation of EGFR, which is associated with unregulated growth and proliferation of tumor cells.8 Modification of the signaling pathways downstream of EGFR in the absence of EGFR alterations is another oncogenic mechanism of tumor cells. Whether the abnormality lies with EGFR or a downstream protein, these EGFR-related signaling cascades contribute to unregulated transcriptional activities related to cell cycle progression, cell proliferation and survival, adhesion, angiogenesis, invasion, and metastasis.4
Given the role of EGFR in colorectal cancer pathogenesis, great interest has developed in recent years surrounding therapies that target EGFR. Cetuximab is a human-mouse chimeric IgG1 monoclonal anti-EGFR antibody that was Food and Drug Administration (FDA)-approved in 2004 as a second-line treatment of colorectal cancer.9 Panitumumab is a fully human IgG2 κ monoclonal antibody that was FDA-approved in the US in 2007 as a third-line monotherapy of refractory colorectal cancer.10 The initial trials found that only about 10% of patients with metastatic colon cancer responded to cetuximab.5,10,11 These therapies remain extremely expensive, costing approximately $80 to 100,000/year12,13 and have potential significant side effects, particularly dermatologic. Therefore, prediction of response (or nonresponse) to anti-EGFR therapies is desirable to avoid unwarranted side effects, costs, and delays in more effective therapy.
Use of EGFR as a biomarker to predict response to EGFR monoclonal antibodies has been largely unsuccessful. EGFR mutations are rare events in colon cancers, occurring in <1%,14,15 and these have not be found to be predictive of anti-EGFR response.16 The amount of EGFR expression measured by immunohistochemistry,5,17 similarly, has not been shown to predict clinical response. Even tumors without any EGFR expression have similar response rates to those with EGFR expression,18,19 and EGFR immunohistochemistry has been abandoned as a determinant of whether patients are eligible for anti-EGFR therapy. EGFR copy number, although promising, has not been widely accepted as a predictor of response to anti-EGFR therapy.15,20 So, the search for predictive biomarkers extended to downstream signaling components of EGFR.
The KRAS (Kirsten rat sarcoma viral oncogene homolog) gene is a member of the RAS family that encodes a G-protein involved in the coupling of signal transduction from surface receptors, including EGFR, to various intracellular targets.9 RAS proteins cycle between active GTP-bound and inactive GDP-bound configurations. RAS-GTP/GDP levels are tightly regulated by the activities of guanine nucleotide exchange factors and GTPase activating proteins, and disruptions in this balance may lead to tumorigenesis.21 Activating KRAS mutations result in RAS proteins, which are constitutively active due to defective intrinsic GTPase activity and resistance to GTPase activating proteins.22 Therefore, activating mutations in KRAS leads to stimulation of the RAS/MAPK signaling pathway independent of EGFR with enhanced cell growth, proliferation, and survival. Thirty percent to 40% of colorectal cancers contain a mutated KRAS oncogene,23,24 and over 3000 point mutations have been reported.8 The most common mutations occur in codons 12 and 13 of exon 2, with approximately 80% occurring in codon 12% and 20% in codon 13.25,26 Activating mutations in codons 61 and 146 have also been reported, but these make up <10% of mutations.9,25 As activating KRAS mutations lead to EGFR independence, it stands to reason that tumors with activating mutations in the KRAS gene will not benefit from anti-EGFR therapies, and this has been well-established in numerous randomized controlled trials and single-arm trials.27,28
In comparison with KRAS wild type tumors, several retrospective single-arm series have shown that KRAS mutations in codons 12 and 13 of exon 2 are associated with lack of treatment response (Table 1),15,29–34 and significantly shorter median progression free survival (Table 2)29,31,32,34 in patients treated with cetuximab or panitumumab alone and cetuximab in combination with chemotherapy. Response rates were significantly greater in wild type tumors than in KRAS mutants in all but one of the single-arm series.15 Overall survival data is less compelling, with only 2 series showing significant differences between anti-EGFR treated KRAS wild type and mutated tumors.30,34 The striking differences in response rates and progression free survival prompted the investigation of KRAS mutation status in several larger, randomized controlled trials,35–38 which confirmed the significant differences in response rates35–38 and median progression free survival35,36,38 between patients with wild type KRAS tumors and those with exon 2 KRAS mutations (Tables 1, 2). Overall survival data remained conflicting in the randomized trials, with 1 series not reporting overall survival data of KRAS wild type versus mutant tumors,38 1 series documenting no significant differences,35 and only 1 showing a significant survival advantage of anti-EGFR therapy in patients with KRAS wild type tumors.36 The final series showed a trend toward a survival advantage in KRAS wild type tumors, but statistical calculations were not performed.37
These trials have shown that up to 40% of patients with KRAS wild type colorectal cancer show at least a partial response to anti-EGFR therapy, an improvement over the 10% before KRAS mutation stratification5,11; whereas, patients with KRAS mutated tumors gain no benefit from anti-EGFR therapy. The utility of determining KRAS mutation status, therefore, is to determine those who will not respond to EGFR monoclonal antibody therapy. Unfortunately, at least 60% of KRAS wild type patients will derive no benefit from anti-EGFR antibodies, highlighting the need for additional biomarkers to help separate responders from nonresponders. Potential biomarkers include BRAF,29,32,39 KRAS codons 61 and 146, PI3K,40–42 and PTEN41,43 mutations and EGFR gene copy number.15,20,39
The National Comprehensive Cancer Network and American Society of Clinical Oncology guidelines recommend that all tumors with metastases should be tested for mutations in codons 12 and 13 of exon 2 of the KRAS gene before initiating anti-EGFR therapy.27,28 There are no FDA approved KRAS mutation tests on the market, and there are no specific methodology recommendations.22 Decisions about whether to institute reflex testing, at least in a subset of cases, are best made through collaboration with all members of the treatment team, and these will likely be institution-specific. At our institution, we reflexively test all stage III and IV colorectal adenocarcinomas; that is, testing is ordered by the pathologist in tumors with lymph node or distant metastases. There may be a time in the near future, however, when it becomes the standard of care for all colorectal carcinomas to undergo KRAS mutational analysis.
As 20% of patients with colorectal carcinoma present with metastatic disease and an additional 30% to 40% develop metastasis during their disease course,44 there are times in which testing the primary tumor is impractical or impossible. In fact, some authors have recommended testing the metastasis rather than the primary tumor, primarily because metastatic disease is the trigger for and the target of anti-EGFR therapy.45 In addition, the anti-EGFR efficacy data is often based on KRAS mutation testing from the metastasis, when specified in the study methods. Concordance rates between the primary tumor and metastases have been found to be >90% in several published series (Table 3).45–51 Although not substantiated, one series documented only 68% concordance between primary site and lymph node metastases52; however, 10/28 (36%) of the cases in that series showed concomitant BRAF and KRAS mutations, and, as these mutations are generally considered mutually exclusive,53 the validity of the overall results may also be in question. In addition, Molinari et al45 and Zauber et al48 found complete concordance in KRAS mutation status in all of their matched primary tumors and lymph node metastases.
Studies specifically addressing KRAS mutation status before and after chemotherapy or radiation are lacking and needed. Nevertheless, previous chemotherapy does not seem to impact the KRAS mutation status of a tumor. All of the available efficacy studies comparing KRAS mutated and wild type tumors have examined anti-EGFR therapies after failing at least one chemotherapy regimen with KRAS mutation testing often performed on the metastasis. Tumor cell density, however, may be insufficient in treated tumors and be a source of false KRAS wild type results, and pretreatment samples often prove invaluable in these instances.
METHODS FOR KRAS MUTATION IDENTIFICATION IN CLINICAL SAMPLES
General Principles of Solid Tumor Molecular Testing
Molecular testing in solid tumors has become standard practice in several different organ systems. The best-developed clinical molecular tests exist in hematopathology and soft tissue pathology, where translocation analysis is a critical diagnostic tool and has a role in disease monitoring. Colorectal carcinoma is another important target for molecular testing with the implementation of microsatellite instability assays for identification of patients at risk for hereditary disease. Newer molecular anatomic pathology tests are focusing on mutation detection as a predictor for response to therapy. The initial studies in lung cancer found that EGFR mutations were associated with response to EGFR inhibitor therapies and were very promising.54–56 Despite that these assays have not become standard practice in most clinical situations, there is great interest in identifying mutations that correlate with response to therapy, particularly in the setting of novel targeted therapies. KRAS in colorectal carcinoma is an example of an assay that has become clinically applicable over a very short period of time.
In molecular anatomic pathology testing, working with paraffin embedded tissues is different than working with fresh or frozen tissue. Assays in molecular anatomic pathology require a design that takes the starting material into careful consideration. The most important general factors that will impact on assay design in solid tumors testing are tumor purity, fixation and processing of tissues, and tumor quantity. Although these are general principles in molecular anatomic pathology, they are especially important for KRAS mutational analysis in colorectal carcinoma cases.
Tumor purity is essential in most molecular anatomic pathology testing because false negatives will be common when the sample is contaminated by high levels of nontumor elements. Microdissection is an excellent way in which to enhance tumor purity.57 Many laboratories require that a sample be at least 75% tumor for testing accuracy. Colorectal carcinoma, in particular, may harbor significant amounts of supporting stroma, necrotic debris, noninvasive neoplastic components, and inflammatory cell infiltrates. For this reason, it is optimal that these samples undergo careful microdissection before extraction of nucleic acids. Using standard manual microdissection, which is easy and inexpensive to perform, tumor enrichment can be accomplished to very high levels, thereby limiting the potential for false negatives and polymerase chain reaction (PCR) inhibition.57
The type of fixative used is another particularly important concern, as some fixatives do not support molecular testing (most notably, picric acid containing solutions, and decalcification solutions).58 Tissues fixed in most standard formalin based fixatives are amenable to molecular testing. But, even in optimally processed tissue that is fixed in formalin, there will be a substantial degree of DNA degradation due to the cross-linking of proteins. Additionally, the time spent in fixative is a critical issue. Tissue fixed for longer than 24 hours has a much lower yield and poorer quality of DNA, which can result in failed molecular testing.58 In any test involving PCR, the assay must have a relatively small PCR product if extracting DNA from paraffin embedded tissue; this helps to overcome the inherent issue of DNA degradation.
Another significant issue in molecular anatomic pathology is the sample size necessary to produce reliable and accurate molecular results. Some assays, such as microsatellite analysis, are known to be unreliable when starting levels of DNA are low.59 Despite that PCR amplification can produce results even from very small samples, high assay failure rates and potential loss of specificity are problematic in these small samples. Different reference laboratories publish different criteria for minimum sample size. Unfortunately, in tissue samples, there may not be an option to obtain larger samples, as there would be in blood-based tests. For this reason, it is our practice to test most samples, regardless of size, but to report small sample size as a potential confounding variable that can affect the sensitivity and specificity of the assay.
One of the most challenging situations in KRAS mutational testing for colorectal carcinoma is performing the analysis on colectomy specimens from patients who have undergone neoadjuvant therapy. In these cases, the tumor cells are often sparse and are embedded and widely distributed in a background of fibrotic or reactive tissues (Fig. 2). These cases present a challenge for microdissection, and the purity of the tumor sample may be at risk. In these cases, we attempt to obtain pretherapy biopsy samples as a better source of DNA for KRAS testing. But, when these samples are not available, we perform careful microdissection under direct visual guidance using a microdissection microscope. Successful amplification is variable in these cases, but we have had reasonable results.
An interesting potential modification to the mutational detection methods discussed below is the use of whole genome amplification (WGA) before analysis for KRAS mutations.60,61 WGA is a method for amplifying the entire DNA contents of a sample nonspecifically to yield more overall starting nucleic acid. It was originally used for preimplantation genetic analysis, where only single cells are available for DNA testing.62 WGA may be a useful adjunctive technique for very small tissue samples that might otherwise be rejected purely on the basis of the size of the tissue biopsy fragments.
Assays for KRAS Mutational Testing
Most assays for detecting point mutations in tumor related samples have either used a method for screening for mutations, direct detection of the mutant gene copy, or a combination of these approaches. The screening methodologies often include conformation based separation techniques, including single strand conformation polymorphism, denaturing gradient gel electrophoresis, and other techniques.63,64 These assays detect the presence of mutants (and polymorphisms), but cannot specifically identify the mutation. The more common techniques involve direct detection of the mutation or combination of prescreening followed by confirmation of the mutation through direct approaches.64
Techniques for direct KRAS gene mutation analysis in colorectal carcinoma have been most widely reported in the literature in 2 basic settings: as part of the testing associated with clinical trials and as techniques for the mutational assays for clinical laboratories. In the former situation, 2 basic methods have predominated. These include dideoxy sequencing, which is the traditional cycle sequencing reaction on the basis of the Sanger method of gene sequencing, and allele specific PCR. Both of these assays are described in detail below, along with discussion of the potential pitfalls in routine clinical testing.
Traditional Gene Sequencing
In the early 1970s, Dr Frederick Sanger developed a method for rapid gene sequencing,65 for which he won the Nobel Prize in Chemistry in 1980.66,67 The specifics of the technique have been modified over the years, but the fundamentals and the basic method remain essentially the same as in those initial experiments. Today, we capitalize on extensive automation, enabling the sequence to be generated quickly and accurately.
In the cycle sequencing reaction, the first step is to obtain DNA from the sample, through standard nucleic acid extraction techniques. The DNA is then subjected to PCR, using primers which are specific for the genomic area being assayed. In the case of KRAS gene mutation analysis, this includes exon 2 of the gene in most clinical assays. This PCR amplicon is then used in the cycle sequencing reaction with one primer, specially formulated nucleotide mixtures, DNA polymerase and other reagents. The primer can either be the same forward or reverse primer from the original PCR reaction, or it can be one that is internally located to the initial primers. The nucleotides mixture contains specially formulated nucleotides that have fluorescent tags and contain a subset of modified nucleotides that are “chain terminating inhibitors of DNA polyermase.65” When these modified nucleotides are incorporated randomly into the product, the DNA polymerase cannot extend and the reaction with that particular amplicon is terminated. The product of the cycle sequencing reaction is loaded into a capillary electrophoresis machine and the sequence is read by analysis of the fluorescent tag that ends each different amplicon.
In the setting of KRAS mutations, there are 2 codons that can harbor mutations (codons 12 and 13) in any of 6 nucleotides. Codons 61 and 146 also have mutations in a minority of colorectal carcinomas.9 The sequence for these specific areas is examined to identify potential mutants. In the electropherogram output from the sequencing reaction, a positive sample yields 2 peaks for a point mutation, one corresponding to the preserved normal allele and one corresponding to the mutant allele (Fig. 3).
Sequencing has long been considered to be the gold standard for detecting point mutations. It is a highly specific technique, with a very low false positive rate. The false positive rate can be even further reduced by performing duplicate sequencing that includes sequencing in both the forward and the reverse directions or by duplicate sample testing.
The major pitfall in traditional sequencing is that it is not very sensitive. This reaction will generally require at least 20% to 25% mutant cells to be present in a background of cells with normal DNA to be able to detect the mutation in the sequence.68 Again, in colorectal carcinoma clinical samples, this type of tumor purity may be difficult to accomplish without microdissection.
Allele Specific Polymerase Chain Reaction
The other method that is frequently reported for KRAS gene sequencing is allele specific PCR. In this method, typical PCR reactions are performed, again starting with DNA obtained from the tumor sample. The PCR primers, however, are designed with the forward primer overlapping codons 12 and 13, where the mutations occur. A set of forward primers is designed to correspond to each of the potential DNA sequences that will be present in mutant cases. The reaction conditions must be selected to have high fidelity and to have little potential for nonspecific binding of the primers. The primers can be fluorescently labeled and analyzed by capillary electrophoresis and fragment analysis for the presence of mutations. Amplification products with only the normal peak present would have no mutation and products with 2 peaks present (1 mutant and 1 normal) would be classified as having a mutation present. Allele specific PCR requires either the performance of multiple different PCR reactions or the multiplexing of the primer sets into one reaction tube. Despite the obvious advantages to decreasing workload and reagents, multiplexing this type of complex mixture can be quite challenging and can lead to nonspecific results.
The major pitfall in allele specific PCR is in the original design of the assay. Most allele specific PCR clinical applications only test for a subset of the most common mutations in colorectal carcinoma, usually with a total of 7 different mutations detected. This will lead to false negatives in a small minority of cases where alternative mutations are present. However, the advantage of allele specific PCR is its very high sensitivity. It is generally thought that this type of reaction will detect tumor mutations at 5% or even lower of the original starting sample. There are several commercially available test kits for KRAS mutation detection that use allele specific PCR. Currently, however, these are not FDA approved and are only available for research purpose in the United States.
Alternative Methods for Mutation Detection
A number of newer technologies have also been applied to testing for KRAS gene mutations.69–73 Some of these may have promise for clinical use in the future. However, others require specialized equipment or procedures that may not be commonly available in most laboratories. In analyzing any of these newer methods, the main concerns that one should address are the sensitivity and the specificity of the assay.74
One of the most promising new techniques is pyrosequencing, which does not rely on electrophoresis, but is a nucleotide extension based technique for gene sequencing.75 This technique can read sequence lengths up to 40 or 50 basepairs.76,77 The technique does require a special instrument (Biotage AB and Biosystems, Uppsala, Sweden), but the machine is relatively inexpensive and may have other applications in the clinical laboratory. The most promising aspects to the assay are the high specificity and high sensitivity and the potential for relatively quick turnaround time for results. In one recent report of a pyrosequencing assay for KRAS mutations, the sensitivity was between 3% and 5%.68 This high sensitivity makes the assay particularly valuable for the typical mixed samples that are found in paraffin embedded tissues.
REPORTING OF RESULTS
There is no consensus on how KRAS mutational results should be reported for colorectal carcinoma. How detailed should the methods description be in clinical reports? Should potential confounding variables be reported? Are there formats that should be used for incorporation of molecular results into anatomic pathology reports?
Most of these general questions related to reporting of molecular results have been addressed in an excellent guideline document.78 The basic components of any molecular report should include the results and interpretation and the methodology used. References could be included for published performance characteristics, or in-house validation data can be included to address potential confounding situations.
One unique question that arises in terms of KRAS mutational assays is whether there is a need to report the exact mutation or just to report the fact that there is a mutation present. There is no current indication in the literature suggesting that different mutations have different impact on therapeutic response, and, therefore, one could argue that it is not necessary to report the specific KRAS mutation. However, there may be a role for knowing what mutation is present in these samples, particularly if evidence of differences in behavior are eventually uncovered. Another potential role would be in follow-up studies of the same patient where molecular testing may play a role for assessing metastatic disease.
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