Review
KRAS mutation testing in clinical practice Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Korea University on 01/09/15 For personal use only.
Expert Rev. Mol. Diagn. Early online, 1–10 (2014)
Sudhir Perincheri and Pei Hui* Department of Pathology, Yale University School of Medicine, 310 Cedar Street, New Haven, CT 06520-8023, USA *Author for correspondence: [email protected]
Activating mutation of KRAS plays a significant role in the pathogenesis of common human malignancies and molecular testing of KRAS mutation has emerged as an essential biomarker in the current practice of clinical oncology. The presence of KRAS mutation is generally associated with clinical aggressiveness of the cancer and reduced survival of the patient. Therapeutically, KRAS mutation testing has maximum utility in stratifying metastatic colorectal carcinoma and lung cancer patients for treatment with targeted therapy. Diagnostically, KRAS mutation testing is useful in the workup of pancreaticobiliary and thyroid cancers, particularly using cytological specimens. In the era of precision medicine, the role of KRAS mutation testing is poised to expand, likely in a setting of combinatorial therapeutic strategy and requiring additional mutation testing of its upstream and/or downstream effectors. KEYWORDS: clinical oncology • KRAS • mutation testing
The role of RAS proteins in tumorigenesis has been studied and characterized for more than four decades [1]. Of the three highly related RAS proteins NRAS, KRAS and HRAS, KRAS has a disproportionate role in cancer pathogenesis across various organ systems. Currently, the COSMIC database catalogs 234 different KRAS mutations that have been characterized in tumors in 42 different tissues [2]. Even though targeted therapies directly against KRAS have proven unsuccessful to date, characterization of KRAS mutations has become critical in the diagnosis of certain cancers and the stratification of patients with common malignancies for which targeted therapies against key oncogenic molecules are increasingly possible [3]. As more targeted therapies become available and combinatorial treatments take center stage in cancer therapy, KRAS mutation testing has become a very important tool in the cancer treatment armamentarium. In this review, we briefly discuss the molecular genetics and biology of KRAS, the various methodologies used for KRAS mutation testing and its clinical applications in the context of specific tumor types for prognosis and treatment. Molecular genetics & biology
The discovery of the RAS family of proteins can be traced back to the 1960s during the isolation of murine leukemia viruses by serial informahealthcare.com
10.1586/14737159.2015.986102
passage in mice and rats [4]. It was later shown that these retroviruses were recombinants carrying rat cellular genes and their role in cellular transformation was shown by the isolation of RAS oncogenes from tumor cell lines and chemically transformed cell lines [5–8]. Definitive proof of their role in carcinogenesis was provided by the identification of mutant KRAS in the biopsy of a lung carcinoma patient that was absent from his white blood cells and his normal lung parenchyma [9]. It is currently estimated that RAS mutations, the majority of which target hotspots in codons 12, 13 and 61, are associated with up to 20–30% of all human cancers [10]. KRAS mutations account for a disproportionate majority of RAS mutations in cancer and have been identified in pancreatic, colon, lung, endometrial and thyroid carcinoma among others (TABLE 1) [11]. KRAS or KRAS2 derives its name from being the homolog of the Kirsten Rat Sarcoma virus and maps to the human chromosome 12p12.1 [12]. Alternative splicing of the KRAS pre-mRNA results in the formation of two transcripts, a minor KRAS2A transcript and the more ubiquitously expressed KRAS2B transcript that code for 188 amino acid and 189 amino acid proteins, respectively [13]. KRAS is essential for survival; KRAS deficient mice die at embryonic day 12–14 due to anemia and defective fetal liver erythropoiesis [14,15]. KRAS and the highly related HRAS
2014 Informa UK Ltd
ISSN 1473-7159
1
Review
Perincheri & Hui
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Korea University on 01/09/15 For personal use only.
Table 1. Frequency of KRAS mutations in common cancers. Cancer
Frequency (%)
Common point mutations
Pancreas
58.6
35G>A, 35G>T, 34G>C
Colorectal
34.8
35G>A, 35G>T, 38G>A
Biliary tract
25.2
35G>A, 35G>T, 34G>A
Small intestine
19.5
35G>A, 35G>T, 38G>A
Lung
16.8
34G>T, 35G>A, 35G>T
Ovary
12.4
35G>A, 35G>T
Endometrium
15.7
35G>A, 35G>T, 35G>C
Sarcomas
4.6
35G>A, 35G>T, 34G>T
Based on data from the COSMIC database.
and NRAS genes, encode a family of 21 kD GTPases that couple extracellular receptor activation to intracellular signaling pathways (FIGURE 1). The GTPases cycle between a GTP-bound ‘on’ state and a GDP-bound ‘off’ state [16,17]. Activation or ligand binding of the extracellular receptors leads to formation of intracellular signaling complexes consisting of docking proteins and guanidine nucleotide exchange factors (GEFs). The GEFs catalyze the removal of GDP from RAS proteins and allow passive binding to GTP. The GTP-bound active RAS can then activate a plethora of downstream pathways including the MAP kinase pathway leading to increased cell proliferation, the PI3K pathway leading to increased survival, activation of signaling proteins such as protein kinase C members affecting calcium signaling and RAC protein leading to altered actin dynamics [10,16]. Activation of KRAS stimulates RalGDS proteins that activate small GTPases such as RalB, which in turn activate TBK1. Activated TBK1 is involved in cytokine production and cell cycle progression through the stimulation of NF-kB and activation of IRF3 and IRF7 [18]. In the physiological state, the RAS signaling is downregulated by GTPaseactivating proteins (GAPs) that increase the intrinsic GTPase activity of RAS proteins many fold causing GTP hydrolysis and the formation of inactive GDP-RAS complex. The first 165 amino acids of the RAS proteins which constitute their G domain are highly conserved among all family members; the remaining C-terminal portion shows considerable variation across members, and is specified as the hypervariable domain [11,16]. The G domain contains epitopes responsible for phosphate and guanine nucleotide ring binding. It also contains protein loops called switch region I (AA30–38) and switch region II (AA59–67) that undergo structural changes upon GTP binding and form interaction surfaces with effectors in a GTP-dependent manner. Crystal structure studies of the RASGDP-GAP complex have shown that the GTP hydrolysis step relies on the insertion of an ‘arginine finger’ by the RasGAP near the b and g phosphates of GTP resulting in neutralization of a negative charge necessary for GTP hydrolysis [19]. The common doi: 10.1586/14737159.2015.986102
oncogenic RAS mutations targeting 12G and 13G introduce amino acids with side chains that interfere with this process causing accumulation of the GTP-bound ‘on’ RAS protein. The third most common oncogenic mutation targeting Q61 in the switch II region interferes with the activation of water molecule necessary for GTP hydrolysis [20]. These mutations thus lead to accumulation of GTP-bound active RAS resulting in uncontrolled activation of downstream pathways. Phenotypically, this results in independence from extracellular growth and inhibitory signals, increased proliferation and decreased apoptosis; all of which are requirements for tumorigenesis. The hypervariable C-terminal region, while less essential for enzymatic activity, undergoes post-translational modification that is necessary for the subcellular localization of RAS proteins. All RAS proteins undergo farnelysation at the C-terminus at a CAAX motif, where C denotes a cysteine, while A stands for an aliphatic amino acid and X is any amino acid [21,22]. HRAS, NRAS and KRAS2A undergo further palmitoylation of cysteine residues at the C-terminus that anchors them to the cell membrane [23,24]. In contrast, KRAS2B lacks cysteine residues at the C-terminus and instead utilizes a polybasic stretch of lysine residues that interact with the negatively charged lipid head groups for cellular localization [25]. It has been argued that this differential localization of KRAS2B and its different microcellular environment may account, at least partly, for the overrepresentation of KRAS mutations in human cancers despite its functional similarity to the other RAS proteins [11]. Lab testing for KRAS mutation
Molecular testing of KRAS mutation is currently employed both for diagnosis (e.g., for pancreatic carcinoma using brushing sample) and for predicting response to targeted therapy (e.g., response to anti-EGFR therapies of metastatic colon cancer) [26]. The starting material for the assays includes formalinfixed paraffin-embedded (FFPE) tissue, snap frozen tissue and cytological preparations. Significant challenges in KRAS testing include limited starting material in cytology and biopsy specimens and the fragmented nature of the DNA isolated from FFPE specimens. Treatment effect from chemoradiation can also significantly reduce the amount of tumor cells. Additional challenges include the presence of heterozygosity of the mutation, the wide spectrum of mutation types, genetic heterogeneity of the tumor and the contribution of wild-type KRAS sequences from the background normal tissue [3,27]. To increase the sensitivity of the tests, often a trained pathologist selects a tumor-enriched area from histological sections for macro- or microdissection before DNA extraction. Currently, essentially all methods utilize PCR with specific primers to amplify mutation hotspots of the KRAS gene (exons 2 and 3) and then employ different downstream techniques to differentiate wildtype sequences from mutant sequences. Common techniques include methods such as Sanger sequencing, pyrosequencing, single-strand conformation polymorphism analysis (SSCP), real-time PCR with melting curve analysis and allele-specific PCR [26,27], which are briefly discussed below. Expert Rev. Mol. Diagn.
KRAS mutation testing
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Korea University on 01/09/15 For personal use only.
Sanger sequencing
Sanger sequencing of PCR-amplified DNA templates is regarded as the gold standard for KRAS testing. Sanger sequencing utilizes a mixture of deoxynucleoside triphosphates (dNTPs) and fluorescently labeled dideoxynucleoside triphosphates (ddNTPs) to generate nested fragments by chain termination during the synthesis of complimentary DNA [28]. The amplified fragments are then separated by high-resolution gel electrophoresis and the sequence is read as the fragments pass through a laser detection system. This method can provide good sequence reads of more than 500 bases, which are sufficient for KRAS mutation testing (FIGURE 2). Sanger sequencing has the advantage that it provides the entire amplified sequence and can therefore identify all possible base mutations in addition to small insertions and deletions. However, it has a low sensitivity or limit of detection of around 15–30% of mutant DNA sequence (defined as the percentage of mutant DNA to wild-type DNA) [29]. A study using automated Sanger sequencing for KRAS testing found that it had low sensitivity for a subset of codon 12 mutations in addition to an 11.1% falsepositive rate and a 6.1% false-negative rate, particularly when testing FFPE tissue samples [29]. Sanger sequencing tends to have greater utility as a confirmatory test for results obtained from other techniques that do not identify definitive specific base changes. Pyrosequencing
In pyrosequencing, DNA polymerase synthesis of complimentary DNA of a solid-support immobilized template DNA is performed with the addition of limiting amounts of specific dNTPs [28]. The inorganic pyrophosphate released after the incorporation of a complimentary base undergoes a series of enzymatic reactions ultimately resulting in a light signal that is detected as a peak in a pyrogram. In this manner, the pyrogram lists the order of incorporated complementary DNA bases and therefore the underlying DNA sequence. While pyrosequencing has been shown to have a detection limit of A) or G12V (35G>T) mutations, 15–20% are G13D mutations (38G>A) [26]. KRAS mutation status has a strong predictive value in colon cancer [47]. Investigations into the prognostic role of KRAS in colon cancer have also suggested that the G12V mutation is associated with an aggressive phenotype and may be associated with reduced overall survival [48,49]. More recent prospective randomized controlled trials have, however, failed to replicate these findings [50,51]. What is indisputable though is the predictive role of KRAS mutation status in colon cancer. The EGF signaling pathway has been increasingly targeted in cancer with the development of monoclonal antibodies and small molecule inhibitors targeting the EGFR pathway. Cetuximab and panitumumab are two anti-EGFR antibodies that have been approved by FDA as first line of treatment of metastatic colon cancer in combination with irinotecan, 5-fluorouracil and leucovorin [47]. Beginning in 2006, a series of retrospective studies were published that showed that KRAS mutations in codons 12 and 13 were negative predictors of response to anti-EGFR therapies [52–59]. Two multicenter Phase III randomized controlled trials later confirmed these findings with both trials showing better progression-free survival and overall survival with anti-EGFR therapy in patients with wild-type KRAS compared with those with mutant KRAS [57,60]. These results prompted the American Society of Clinical Oncology and the National Comprehensive Cancer Network (NCCN) to issue guidelines that recommended the KRAS mutation status (at least codons 12 and 13) be assessed prior to EGFR therapy [61–63]. Subsequently, the PRIME study demonstrated that panitumumab-FOLFOX4 was well tolerated and informahealthcare.com
Review
significantly improved progression-free survival in patients with wild-type KRAS tumors and underscores the importance of KRAS testing for patients with metastatic colorectal carcinoma [64]. The Phase III FIRE-3 Trial Data confirmed that most patients with wild-type RAS metastatic colorectal cancer benefited from first-line irinotecan, 5-fluorouracil and leucovorin plus cetuximab treatment [65]. Because of the good correlation of KRAS mutation status between the primary site and metastatic cancer, this testing can be performed on archival tissue from the original biopsy if necessary [66]. More recent studies have shown that non-exon 2 mutations are also associated with failure of response to EGFR therapy [67]. The picture has been complicated somewhat by another retrospective study that documented a positive association between G13D mutation of KRAS codon 13 and response to cetuximab, showing that the types of KRAS mutation may also be important in determining response to anti-EGFR therapy [68]. It is to be noted that KRAS mutations account for less than 40% of anti-EGFR refractory colon cancers. Mutations in BRAF, PIK3CA and NRAS and loss of PTEN expression account for a significant proportion of wild-type KRAS cases that fail to respond to anti-EGFR therapy [47]. In its updated guidelines, NCCN now recommends KRAS and NRAS testing in colon cancer. Moreover, it has been suggested by some investigators that testing for KRAS, BRAF, NRAS, PIK3CA, PTEN and MSI are necessary to identify the patients who most likely benefit from antiEGFR therapy [47]. Lung cancer
KRAS mutations are the most common somatic mutations in non-small-cell lung cancer (NSCLC), accounting for approximately 20% of all mutations with 63 documented different mutations in the COSMIC database [2]. They are strongly associated with a history of smoking, are found more commonly in adenocarcinomas and rarely in squamous cell carcinomas and are seen less commonly in Asians compared with Caucasians [69]. As in colon cancer, the majority of mutations are missense mutation involving exon 2 with the G12C mutation being more frequent. The predictive significance of KRAS mutation in NSLC is questionable since multiple studies have failed to show any association between KRAS mutations and response to conventional chemotherapy [69]. With the FDA approval of small molecule EGFR inhibitors like erlotinib and gefitinib as second- and third-line treatment of NSCLC, it is of great interest to ascertain if KRAS mutation status in NSCLC has the same predictive value for response to antiEGFR therapy as in colon cancer. Studies to date have shown that the EGFR mutation status has a higher predictive value for this purpose [70–72]. However, given that KRAS mutations are mutually exclusive to EGFR mutation and chromosomal rearrangement of ALK and ROS, the utility of KRAS mutation testing in NSCLC may lie in ruling out the other mutations in a screening algorithm. Currently, the NCCN guidelines make specific recommendations for KRAS mutation testing in NSCLC. doi: 10.1586/14737159.2015.986102
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Korea University on 01/09/15 For personal use only.
Review
Perincheri & Hui
Pancreatic & biliary cancer
KRAS mutation in other human cancers
KRAS mutations are seen in 70–90% of pancreatic ductal carcinomas and 31% of biliary tract carcinomas, respectively [73]. The majority of the mutations are again seen in exon 2, with 35G>A (G12D) being the most common [2]. KRAS mutation testing has a complimentary role in the diagnostic work-up of cytology specimens either from endoscopic retrograde cholangiopancreatography brushings or fine-needle aspiration specimens of pancreatic cystic fluid [74–76]. The presence of KRAS mutation has a high predictive value (94%) for malignancy in patients with indeterminate pancreaticobiliary cytology and should be included as an important adjuvant diagnostic marker [77]. However, molecular analysis alone has low sensitivity in the diagnosis of these lesions. KRAS, in association with cytology and loss of heterozygosity analysis, can increase both the sensitivity and positive predictive value in the diagnosis of malignant pancreatobiliary lesions [74].
In addition to the above malignancies, KRAS mutations have been documented in soft tissue, breast, esophageal, prostate and hematological malignancies among others, although the frequencies of the mutation is generally below 5% in these malignancies. KRAS mutation has been characterized in a case of imatinib-resistant chronic myelogenous leukemia [87]. KRAS mutation testing may be clinically useful in all such situations.
Thyroid carcinoma
KRAS mutations are found with varying frequencies in the various histological subtypes of thyroid carcinoma. While it is not as specific as BRAF mutation for papillary thyroid carcinoma, RAS mutations are seen in approximately 10–20% of papillary thyroid carcinomas particularly of the follicular variant, up to 50% of follicular carcinomas and have a positive predictive value of 74–88% for malignancy [78]. The mutations again tend to be missense mutations clustered in exon 2 with a few affecting Q61 [2]. However, RAS mutations are not highly specific for malignancy since they may be detectable in follicular adenomas. RAS mutations may correlate with more aggressive behavior of thyroid carcinomas [79], implying necessity for total thyroidectomy in lesions harboring them. Female genital tract tumors
KRAS mutations targeting exon 2 are seen more commonly in type 1 ovarian epithelial carcinomas including low-grade serous carcinoma and borderline tumors of the ovary. They are more often associated with an indolent course and better prognosis compared with type 2 ovarian epithelial tumors that harbor p53 mutations [80,81]. More recently, a variant allele in the KRAS-3´ untranslated region called the KRAS-variant has been described that is present in 61% of cases of hereditary breast and ovarian carcinoma syndrome [82]. This mutation alters a let-7 miRNA binding site in the KRAS-3´ untranslated region leading to deregulated levels of KRAS with low levels of background let-7 miRNA. KRAS mutations have also been detected in endometrial mucinous lesions [83,84] and have been linked to the pathogenesis to type I endometrial carcinomas that tend to be low grade with endometrioid morphology [85] and a favorable prognosis. Moreover, a recent study has shown that the nature of KRAS mutation may affect tumor behavior. In this study, tumors with KRAS amplification have a worse behavior than tumors with KRAS point mutations, again underlying the different mechanisms by which KRAS can influence tumor behavior [86]. doi: 10.1586/14737159.2015.986102
Expert commentary
KRAS mutation testing has significant clinical applications in both diagnostic and predictive scenarios. Currently, it has maximum utility in stratifying metastatic colorectal carcinoma patients for treatment with EGFR inhibitors and in diagnostic work-up of cytology specimens. KRAS testing has more of an adjunct role in the diagnostic work-up of thyroid nodules and pancreatic lesions, where the results should be interpreted in the context of histopathologic findings along with clinical and imaging data. The methodologies used in molecular diagnostic laboratories are currently dictated by the clinical specimen types and technical resources available; however, they are usually within the purview of most tertiary medical centers. Five-year view
KRAS has emerged as an essential molecular marker in the clinical practice of oncology. While molecular testing for KRAS mutation is primarily used for stratifying patients for antiEGFR therapy of colorectal and lung cancers at current time, its role is likely to expand in the near future. Numerous clinical trials exploring a plethora of small molecules for precision cancer therapies are likely to open many more venues for KRAS testing, likely in a setting of combinatorial therapeutic strategy and requiring not only characterizing KRAS mutations, but also identifying mutations of both upstream and downstream effectors. The trend is most visible already in the treatment of metastatic colorectal cancer where BRAF and NRAS testing are recommended along with KRAS. Novel technologies such as non-invasive blood-based assays to analyze circulating DNA for somatic mutations [88], digital PCR [89,90], Sequenom (a mass spectrometry genotyping assay that is suitable for broad mutation screening) [91] and the advent of nextgeneration sequencing are poised to further refine the landscape of clinical KRAS mutation analysis. Notwithstanding the challenges of working with FFPE-extracted DNA, nextgeneration sequencing can be used on archival tissue and has the potential to generate more actionable data. Next-generation sequencing is capable of increasing the clinical sensitivity of the test without decreasing the specificity of the analysis [92]. It will likely replace Sanger sequencing as the reference technique for diagnostic detection of KRAS mutation in archival tumor tissues [93]. On the other hand, tumor heterogeneity and the technical expertise needed to extract clinically important mutations from background noise will likely keep it a niche Expert Rev. Mol. Diagn.
KRAS mutation testing
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Korea University on 01/09/15 For personal use only.
expertise confined to specialized labs. A probable scenario that can be envisaged is one in which local molecular diagnostic labs perform routine PCR-based assays for screening purposes as these will, in theory, capture the vast majority of driver mutations, such as mutant KRAS. Once these are confirmed negative, the specimens may then be triaged to a reference lab for additional esoteric testing. Cancer molecular testing is a rapidly evolving and challenging field for which, in the era of precision medicine, the next decade is likely to be more exciting and rewarding.
Review
Acknowledgement
The authors thank M Talmor for her outstanding technical assistance in the preparation of this work. Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.
Key issues • Activating mutations in the RAS family GTPases that lock the RAS proteins in the GTP bound ‘on’ state are associated with 20–30% of all human malignancies with KRAS mutations accounting for a disproportionate majority. • A variety of molecular methods including Sanger sequencing, high-resolution melting analysis, single-strand conformation polymorphism analysis and allele-specific PCR methods that target mutation hotspots in KRAS exons 2 and 3 are currently employed in clinical testing of KRAS mutations and have shown good general concordance in comparative studies. • National Comprehensive Cancer Network guidelines recommend KRAS mutation testing in the clinical work-up of metastatic colorectal cancer where multiple clinical trials have shown that wild-type KRAS status is associated with a better response to anti-EGFR therapy. • Diagnostically, KRAS mutation is useful in the work-up of cytology specimens like pancreatobiliary brush specimens and thyroid fineneedle aspiration material where it can increase the positive predictive value for malignancy. • Novel methods such as next-generation sequencing and mass spectrometry genotyping assays have been shown to increase clinical sensitivity of KRAS mutation testing in clinical samples and are likely to replace current methods in the era of targeted combinatorial therapy.
References
6.
Scolnick EM, Rands E, Williams D, Parks WP. Studies on the nucleic acid sequences of Kirsten sarcoma virus: a model for formation of a mammalian RNA-containing sarcoma virus. J Virol 1973;12(3):458-63
7.
Perucho M, Goldfarb M, Shimizu K, et al. Human-tumor-derived cell lines contain common and different transforming genes. Cell 1981;27(3 Pt 2):467-76
8.
Shih C, Shilo BZ, Goldfarb MP, et al. Passage of phenotypes of chemically transformed cells via transfection of DNA and chromatin. Proc Natl Acad Sci USA 1979;76(11):5714-18
Papers of special note have been highlighted as: • of interest •• of considerable interest 1.
•
Malumbres M, Barbacid M. RAS oncogenes: the first 30 years. Nature Rev Cancer 2003;3(6):459-65 A concise summary of the major developments in understanding the biology of RAS oncogenes and their role in disease.
2.
Bamford S, Dawson E, Forbes S, et al. The COSMIC (Catalogue of Somatic Mutations in Cancer) database and website. Br J Cancer 2004;91(2):355-8
3.
Jancik S, Drabek J, Radzioch D, Hajduch M. Clinical relevance of KRAS in human cancers. J Biomed Biotechnol 2010;2010:150960
4.
Kirsten WH, Mayer LA. Morphologic responses to a murine erythroblastosis virus. J Natl Cancer Ins 1967;39(2):311-35
5.
Ellis RW, Defeo D, Shih TY, et al. The p21 src genes of Harvey and Kirsten sarcoma viruses originate from divergent members of a family of normal vertebrate genes. Nature 1981;292(5823):506-11
informahealthcare.com
9.
10.
Santos E, Martin-Zanca D, Reddy EP, et al. Malignant activation of a K-ras oncogene in lung carcinoma but not in normal tissue of the same patient. Science 1984;223(4637): 661-4 Rajalingam K, Schreck R, Rapp UR, Albert S. Ras oncogenes and their downstream targets. Biochim Biophys Acta 2007;1773(8):1177-95
11.
Ellis CA, Clark G. The importance of being K-Ras. Cell Signal 2000;12(7):425-34
12.
Barbacid M. ras genes. Ann Rev Biochem 1987;56:779-827
13.
Plowman SJ, Berry RL, Bader SA, et al. K-ras 4A and 4B are co-expressed widely in human tissues, and their ratio is altered in sporadic colorectal cancer. J Exp Clin Cancer Res 2006;25(2):259-67
14.
Johnson L, Greenbaum D, Cichowski K, et al. K-ras is an essential gene in the mouse with partial functional overlap with N-ras. Genes Dev 1997;11(19):2468-81
15.
Khalaf WF, White H, Wenning MJ, et al. K-Ras is essential for normal fetal liver erythropoiesis. Blood 2005;105(9):3538-41
16.
Schubbert S, Shannon K, Bollag G. Hyperactive Ras in developmental disorders and cancer. Nature Rev Cancer 2007;7(4): 295-308
17.
Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web. Nature Rev Cancer 2011;11(11):761-74
•
A comprehensive review of RAS oncogenes and their role in cancer.
18.
Downward J. Cancer: a tumour gene’s fatal flaws. Nature 2009;462(7269):44-5
19.
Scheffzek K, Ahmadian MR, Kabsch W, et al. The Ras-RasGAP complex: structural basis for GTPase activation and its loss in
doi: 10.1586/14737159.2015.986102
Review
Perincheri & Hui
oncogenic Ras mutants. Science 1997; 277(5324):333-8 ••
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Korea University on 01/09/15 For personal use only.
20.
21.
Outstanding review describing the structural basis of oncogenic RAS mutations. Graham DL, Eccleston JF, Lowe PN. The conserved arginine in rho-GTPase-activating protein is essential for efficient catalysis but not for complex formation with Rho.GDP and aluminum fluoride. Biochemistry 1999; 38(3):985-91 Magee AI, Newman CM, Giannakouros T, et al. Lipid modifications and function of the ras superfamily of proteins. Biochem Soc Trans 1992;20(2):497-9
22.
Cox AD, Der CJ. Protein prenylation: more than just glue? Curr Opin Cell Biol 1992; 4(6):1008-16
23.
Dudler T, Gelb MH. Palmitoylation of Ha-Ras facilitates membrane binding, activation of downstream effectors, and meiotic maturation in Xenopus oocytes. J Biol Chem 1996;271(19):11541-7
24.
25.
Milligan G, Parenti M, Magee AI. The dynamic role of palmitoylation in signal transduction. Trends Biochem Sci 1995; 20(5):181-7 Jackson JH, Li JW, Buss JE, et al. Polylysine domain of K-ras 4B protein is crucial for malignant transformation. Proc Natl Acad Sci USA 1994;91(26):12730-4
26.
Anderson SM. Laboratory methods for KRAS mutation analysis. Expert Rev Mol Diagn 2011;11(6):635-42
27.
Shackelford RE, Whitling NA, McNab P, et al. KRAS Testing: a Tool for the Implementation of Personalized Medicine. Genes Cancer 2012;3(7-8):459-66
28.
Metzker ML. Emerging technologies in DNA sequencing. Genome Res 2005; 15(12):1767-76
29.
Tsiatis AC, Norris-Kirby A, Rich RG, et al. Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications. J Mol Diagn 2010;12(4):425-32
•
Excellent article evaluating the efficacy of various methods for KRAS mutation testing in the clinical setting.
30.
Dufort S, Richard MJ, de Fraipont F. Pyrosequencing method to detect KRAS mutation in formalin-fixed and paraffin-embedded tumor tissues. Analytical Biochem 2009;391(2):166-8
31.
Ogino S, Kawasaki T, Brahmandam M, et al. Sensitive sequencing method for KRAS mutation detection by
doi: 10.1586/14737159.2015.986102
embedded tissue using mutant-enriched PCR and reverse-hybridization. J Mol Diagn 2009;11(6):508-13
Pyrosequencing. J Mol Diagn 2005;7(3): 413-21 32.
Weichert W, Schewe C, Lehmann A, et al. KRAS genotyping of paraffin-embedded colorectal cancer tissue in routine diagnostics: comparison of methods and impact of histology. J Mol Diagn 2010; 12(1):35-42
44.
Whitehall V, Tran K, Umapathy A, et al. A multicenter blinded study to evaluate KRAS mutation testing methodologies in the clinical setting. J Mol Diagn 2009; 11(6):543-52
33.
Gao J, Li YY, Sun PN, Shen L. Comparative analysis of dideoxy sequencing, the KRAS StripAssay and pyrosequencing for detection of KRAS mutation. World J Gastroenterol 2010;16(38):4858-64
45.
Forrester K, Almoguera C, Han K, et al. Detection of high incidence of K-ras oncogenes during human colon tumorigenesis. Nature 1987;327(6120): 298-303
34.
Krypuy M, Newnham GM, Thomas DM, et al. High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer. BMC Cancer 2006;6:295
46.
Vogelstein B, Fearon ER, Hamilton SR, et al. Genetic alterations during colorectal-tumor development. N Engl J Med 1988;319(9):525-32
47.
Tan C, Du X. KRAS mutation testing in metastatic colorectal cancer. World J Gastroenterol 2012;18(37):5171-80
48.
Andreyev HJ, Norman AR, Cunningham D, et al. Kirsten ras mutations in patients with colorectal cancer: the multicenter "RASCAL" study. J Natl Cancer Inst 1998;90(9):675-84
49.
Andreyev HJ, Norman AR, Cunningham D, et al. Kirsten ras mutations in patients with colorectal cancer: the ’RASCAL II’ study. Br J Cancer 2001;85(5):692-6
50.
Roth AD, Tejpar S, Delorenzi M, et al. Prognostic role of KRAS and BRAF in stage II and III resected colon cancer: results of the translational study on the PETACC-3, EORTC 40993, SAKK 60-00 trial. J Clin Oncol 2010;28(3):466-74
51.
Ogino S, Meyerhardt JA, Irahara N, et al. KRAS mutation in stage III colon cancer and clinical outcome following intergroup trial CALGB 89803. Clin Cancer Res 2009; 15(23):7322-9
52.
Lievre A, Bachet JB, Le Corre D, et al. KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer Res 2006;66(8):3992-5
•
First published report of the correlation between KRAS mutation status and response to anti-EGFR therapy.
53.
Khambata-Ford S, Garrett CR, Meropol NJ, et al. Expression of epiregulin and amphiregulin and K-ras mutation status predict disease control in metastatic colorectal cancer patients treated with cetuximab. J Clin Oncol 2007;25(22): 3230-7
54.
Di Fiore F, Blanchard F, Charbonnier F, et al. Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by Cetuximab plus chemotherapy. Br J Cancer 2007;96(8):1166-9
35.
Franklin WA, Haney J, Sugita M, et al. KRAS mutation: comparison of testing methods and tissue sampling techniques in colon cancer. J Mol Diagn 2010;12(1): 43-50
36.
Ziai J, Hui P. BRAF mutation testing in clinical practice. Expert Rev Mol Diagn 2012;12(2):127-38
37.
Emanuel JR, Damico C, Ahn S, et al. Highly sensitive nonradioactive single-strand conformational polymorphism. Detection of Ki-ras mutations. Diagn Mol Pathol B 1996;5(4):260-4
38.
39.
40.
41.
42.
43.
Dillon DA, Johnson CC, Topazian MD, et al. The utility of Ki-ras mutation analysis in the cytologic diagnosis of pancreatobiliary neoplasma. Cancer J 2000;6(5):294-301 Hayes VM, Westra JL, Verlind E, et al. New comprehensive denaturing-gradient-gelelectrophoresis assay for KRAS mutation detection applied to paraffin-embedded tumours. Genes Chromosomes Cancer 2000;29(4):309-14 Oliner K, Juan T, Suggs S, et al. A comparability study of 5 commercial KRAS tests. Diagnostic Pathol 2010;5:23 Pritchard CC, Akagi L, Reddy PL, et al. COLD-PCR enhanced melting curve analysis improves diagnostic accuracy for KRAS mutations in colorectal carcinoma. BMC Clin Pathol 2010;10:6 Kriegshauser G, Auner V, Schuster E, et al. KRAS mutation analysis in genomic DNA isolated from formalin-fixed paraffinembedded ovarian tissue: evaluation of a strip-based reverse-hybridisation assay. J Clin Pathol 2011;64(3):252-6 Ausch C, Buxhofer-Ausch V, Oberkanins C, et al. Sensitive detection of KRAS mutations in archived formalin-fixed paraffin-
Expert Rev. Mol. Diagn.
KRAS mutation testing
55.
56.
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Korea University on 01/09/15 For personal use only.
57.
58.
59.
60.
61.
De Roock W, Piessevaux H, De Schutter J, et al. KRAS wild-type state predicts survival and is associated to early radiological response in metastatic colorectal cancer treated with cetuximab. Ann Oncol 2008; 19(3):508-15 Bibeau F, Frugier H, Boissiere-Michot F. [Responses to targeted therapies: colorectal cancer]. Ann Pathol 2009;29(Spec No 1): S74-6
PRIME study. J Clin Oncol 2010;28(31): 4697-705 65.
66.
Amado RG, Wolf M, Peeters M, et al. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol 2008;26(10):1626-34 Freeman DJ, Juan T, Reiner M, et al. Association of K-ras mutational status and clinical outcomes in patients with metastatic colorectal cancer receiving panitumumab alone. Clin Colorectal Cancer 2008;7(3): 184-90 Adelstein BA, Dobbins TA, Harris CA, et al. A systematic review and meta-analysis of KRAS status as the determinant of response to anti-EGFR antibodies and the impact of partner chemotherapy in metastatic colorectal cancer. Eur J Cancer 2011;47(9):1343-54 Karapetis CS, Khambata-Ford S, Jonker DJ, et al. K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med 2008;359(17):1757-65 Allegra CJ, Jessup JM, Somerfield MR, et al. American Society of Clinical Oncology provisional clinical opinion: testing for KRAS gene mutations in patients with metastatic colorectal carcinoma to predict response to anti-epidermal growth factor receptor monoclonal antibody therapy. J Clin Oncol 2009;27(12):2091-6
••
Excellent article summarizing the evidence behind recommendations for KRAS testing in metastatic colon cancer.
62.
Engstrom PF, Arnoletti JP, Benson AB 3rd, et al. NCCN Clinical Practice Guidelines in Oncology: colon cancer. J Natl Compr Canc Netw 2009;7(8):778-831
63.
Engstrom PF, Arnoletti JP, Benson AB 3rd, et al. NCCN Clinical Practice Guidelines in Oncology: rectal cancer. J Natl Compr Canc Netw 2009;7(8):838-81
64.
Douillard JY, Siena S, Cassidy J, et al. Randomized, phase III trial of panitumumab with infusional fluorouracil, leucovorin, and oxaliplatin (FOLFOX4) versus FOLFOX4 alone as first-line treatment in patients with previously untreated metastatic colorectal cancer: the
informahealthcare.com
Heinemann V, von Weikersthal LF, Decker T, et al. FOLFIRI plus cetuximab versus FOLFIRI plus bevacizumab as first-line treatment for patients with metastatic colorectal cancer (FIRE-3): a randomised, open-label, phase 3 trial. Lancet Oncol 2014;15(10):1065-75 Etienne-Grimaldi MC, Formento JL, Francoual M, et al. K-Ras mutations and treatment outcome in colorectal cancer patients receiving exclusive fluoropyrimidine therapy. Clin Cancer Res 2008;14(15): 4830-5
67.
Douillard JY, Oliner KS, Siena S, et al. Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer. N Engl J Med 2013;369(11):1023-34
68.
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(16):1812-20
69.
Roberts PJ, Stinchcombe TE. KRAS mutation: should we test for it, and does it matter? J Clin Oncol 2013;31(8):1112-21
70.
Zhu CQ, da Cunha Santos G, Ding K, et al. Role of KRAS and EGFR as biomarkers of response to erlotinib in National Cancer Institute of Canada Clinical Trials Group Study BR.21. J Clin Oncol 2008;26(26):4268-75
71.
Brugger W, Triller N, Blasinska-Morawiec M, et al. Prospective molecular marker analyses of EGFR and KRAS from a randomized, placebo-controlled study of erlotinib maintenance therapy in advanced non-small-cell lung cancer. J Clin Oncol 2011;29(31):4113-20
72.
Hirsch FR. K-ras mutations in lung cancer: the "mysterious mutation". Clin Lung Cancer 2006;8(1):11-12
73.
Miglio U, Oldani A, Mezzapelle R, et al. KRAS mutational analysis in ductal adenocarcinoma of the pancreas and its clinical significance. Pathol Res Pract 2014; 210(5):307-11
74.
Somoza AD, Aly FZ. Utility of molecular tests in cytopathology. CytoJournal 2014;11:5
75.
Kipp BR, Fritcher EG, Clayton AC, et al. Comparison of KRAS mutation analysis and FISH for detecting pancreatobiliary tract cancer in cytology specimens collected during endoscopic retrograde
Review
cholangiopancreatography. J Mol Diagn 2010;12(6):780-6 76.
Sturm PD, Rauws EA, Hruban RH, et al. Clinical value of K-ras codon 12 analysis and endobiliary brush cytology for the diagnosis of malignant extrahepatic bile duct stenosis. Clin Cancer Res 1999;5(3):629-35
77.
Cai G, Mahooti S, Lipata FM, et al. Diagnostic value of K-ras mutation analysis for pancreaticobiliary cytology specimens with indeterminate diagnosis. Cancer Cytopathol 2012;120(5):313-18
78.
Nikiforov YE. Molecular diagnostics of thyroid tumors. Arch Pathol Lab Med 2011;135(5):569-77
79.
Garcia-Rostan G, Zhao H, Camp RL, et al. ras mutations are associated with aggressive tumor phenotypes and poor prognosis in thyroid cancer. J Clin Oncol 2003;21(17): 3226-35
80.
Shih Ie M, Kurman RJ. Ovarian tumorigenesis: a proposed model based on morphological and molecular genetic analysis. Am J Pathol 2004;164(5):1511-18
81.
Singer G, Oldt R 3rd, Cohen Y, et al. Mutations in BRAF and KRAS characterize the development of low-grade ovarian serous carcinoma. J Natl Cancer Inst 2003;95(6): 484-6
82.
Ratner E, Lu L, Boeke M, et al. A KRAS-variant in ovarian cancer acts as a genetic marker of cancer risk. Cancer Res 2010;70(16):6509-15
83.
Alomari A, Abi-Raad R, Buza N, Hui P. Frequent KRAS mutation in complex mucinous epithelial lesions of the endometrium. Mod Pathol 2014;27(5): 675-80
84.
Ip PP, Irving JA, McCluggage WG, et al. Papillary proliferation of the endometrium: a clinicopathologic study of 59 cases of simple and complex papillae without cytologic atypia. Am J Surg Pathol 2013; 37(2):167-77
85.
Xiong J, He M, Jackson C, et al. Endometrial carcinomas with significant mucinous differentiation associated with higher frequency of k-ras mutations: a morphologic and molecular correlation study. Int J Gynecol Cancer 2013;23(7): 1231-6
86.
Birkeland E, Wik E, Mjos S, et al. KRAS gene amplification and overexpression but not mutation associates with aggressive and metastatic endometrial cancer. Br J Cancer 2012;107(12):1997-2004
87.
Agarwal A, Eide CA, Harlow A, et al. An activating KRAS mutation in
doi: 10.1586/14737159.2015.986102
Review
Perincheri & Hui
adenocarcinoma. Cancer Biol Ther 2012; 13(10):871-9
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Korea University on 01/09/15 For personal use only.
imatinib-resistant chronic myeloid leukemia. Leukemia 2008;22(12):2269-72 88.
Holdhoff M, Schmidt K, Donehower R, Diaz LA Jr. Analysis of circulating tumor DNA to confirm somatic KRAS mutations. J Natl Cancer Inst 2009;101(18):1284-5
89.
Azuara D, Ginesta MM, Gausachs M, et al. Nanofluidic digital PCR for KRAS mutation detection and quantification in gastrointestinal cancer. Clin Chem 2012; 58(9):1332-41
90.
Zhou B, Irwanto A, Guo YM, et al. Exome sequencing and digital PCR analyses reveal novel mutated genes related to the metastasis of pancreatic ductal
doi: 10.1586/14737159.2015.986102
91.
92.
Arcila M, Lau C, Nafa K, Ladanyi M. Detection of KRAS and BRAF mutations in colorectal carcinoma roles for high-sensitivity locked nucleic acid-PCR sequencing and broad-spectrum mass spectrometry genotyping. J Mol Diagn 2011;13(1):64-73 de Biase D, Visani M, Baccarini P, et al. Next generation sequencing improves the accuracy of KRAS mutation analysis in endoscopic ultrasound fine needle aspiration pancreatic lesions. PLoS One 2014;9(2): e87651
93.
Altimari A, de Biase D, De Maglio G, et al. 454 next generation-sequencing outperforms allele-specific PCR, Sanger sequencing, and pyrosequencing for routine KRAS mutation analysis of formalin-fixed, paraffin-embedded samples. OncoTargets Ther 2013;6:1057-64
••
Excellent review comparing next-generation sequencing with traditional methods of KRAS mutation testing in clinical specimens.
Expert Rev. Mol. Diagn.
KRAS mutation testing in clinical practice Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Korea University on 01/09/15 For personal use only.
Expert Rev. Mol. Diagn. Early online, 1–10 (2014)
Sudhir Perincheri and Pei Hui* Department of Pathology, Yale University School of Medicine, 310 Cedar Street, New Haven, CT 06520-8023, USA *Author for correspondence: [email protected]
Activating mutation of KRAS plays a significant role in the pathogenesis of common human malignancies and molecular testing of KRAS mutation has emerged as an essential biomarker in the current practice of clinical oncology. The presence of KRAS mutation is generally associated with clinical aggressiveness of the cancer and reduced survival of the patient. Therapeutically, KRAS mutation testing has maximum utility in stratifying metastatic colorectal carcinoma and lung cancer patients for treatment with targeted therapy. Diagnostically, KRAS mutation testing is useful in the workup of pancreaticobiliary and thyroid cancers, particularly using cytological specimens. In the era of precision medicine, the role of KRAS mutation testing is poised to expand, likely in a setting of combinatorial therapeutic strategy and requiring additional mutation testing of its upstream and/or downstream effectors. KEYWORDS: clinical oncology • KRAS • mutation testing
The role of RAS proteins in tumorigenesis has been studied and characterized for more than four decades [1]. Of the three highly related RAS proteins NRAS, KRAS and HRAS, KRAS has a disproportionate role in cancer pathogenesis across various organ systems. Currently, the COSMIC database catalogs 234 different KRAS mutations that have been characterized in tumors in 42 different tissues [2]. Even though targeted therapies directly against KRAS have proven unsuccessful to date, characterization of KRAS mutations has become critical in the diagnosis of certain cancers and the stratification of patients with common malignancies for which targeted therapies against key oncogenic molecules are increasingly possible [3]. As more targeted therapies become available and combinatorial treatments take center stage in cancer therapy, KRAS mutation testing has become a very important tool in the cancer treatment armamentarium. In this review, we briefly discuss the molecular genetics and biology of KRAS, the various methodologies used for KRAS mutation testing and its clinical applications in the context of specific tumor types for prognosis and treatment. Molecular genetics & biology
The discovery of the RAS family of proteins can be traced back to the 1960s during the isolation of murine leukemia viruses by serial informahealthcare.com
10.1586/14737159.2015.986102
passage in mice and rats [4]. It was later shown that these retroviruses were recombinants carrying rat cellular genes and their role in cellular transformation was shown by the isolation of RAS oncogenes from tumor cell lines and chemically transformed cell lines [5–8]. Definitive proof of their role in carcinogenesis was provided by the identification of mutant KRAS in the biopsy of a lung carcinoma patient that was absent from his white blood cells and his normal lung parenchyma [9]. It is currently estimated that RAS mutations, the majority of which target hotspots in codons 12, 13 and 61, are associated with up to 20–30% of all human cancers [10]. KRAS mutations account for a disproportionate majority of RAS mutations in cancer and have been identified in pancreatic, colon, lung, endometrial and thyroid carcinoma among others (TABLE 1) [11]. KRAS or KRAS2 derives its name from being the homolog of the Kirsten Rat Sarcoma virus and maps to the human chromosome 12p12.1 [12]. Alternative splicing of the KRAS pre-mRNA results in the formation of two transcripts, a minor KRAS2A transcript and the more ubiquitously expressed KRAS2B transcript that code for 188 amino acid and 189 amino acid proteins, respectively [13]. KRAS is essential for survival; KRAS deficient mice die at embryonic day 12–14 due to anemia and defective fetal liver erythropoiesis [14,15]. KRAS and the highly related HRAS
2014 Informa UK Ltd
ISSN 1473-7159
1
Review
Perincheri & Hui
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Korea University on 01/09/15 For personal use only.
Table 1. Frequency of KRAS mutations in common cancers. Cancer
Frequency (%)
Common point mutations
Pancreas
58.6
35G>A, 35G>T, 34G>C
Colorectal
34.8
35G>A, 35G>T, 38G>A
Biliary tract
25.2
35G>A, 35G>T, 34G>A
Small intestine
19.5
35G>A, 35G>T, 38G>A
Lung
16.8
34G>T, 35G>A, 35G>T
Ovary
12.4
35G>A, 35G>T
Endometrium
15.7
35G>A, 35G>T, 35G>C
Sarcomas
4.6
35G>A, 35G>T, 34G>T
Based on data from the COSMIC database.
and NRAS genes, encode a family of 21 kD GTPases that couple extracellular receptor activation to intracellular signaling pathways (FIGURE 1). The GTPases cycle between a GTP-bound ‘on’ state and a GDP-bound ‘off’ state [16,17]. Activation or ligand binding of the extracellular receptors leads to formation of intracellular signaling complexes consisting of docking proteins and guanidine nucleotide exchange factors (GEFs). The GEFs catalyze the removal of GDP from RAS proteins and allow passive binding to GTP. The GTP-bound active RAS can then activate a plethora of downstream pathways including the MAP kinase pathway leading to increased cell proliferation, the PI3K pathway leading to increased survival, activation of signaling proteins such as protein kinase C members affecting calcium signaling and RAC protein leading to altered actin dynamics [10,16]. Activation of KRAS stimulates RalGDS proteins that activate small GTPases such as RalB, which in turn activate TBK1. Activated TBK1 is involved in cytokine production and cell cycle progression through the stimulation of NF-kB and activation of IRF3 and IRF7 [18]. In the physiological state, the RAS signaling is downregulated by GTPaseactivating proteins (GAPs) that increase the intrinsic GTPase activity of RAS proteins many fold causing GTP hydrolysis and the formation of inactive GDP-RAS complex. The first 165 amino acids of the RAS proteins which constitute their G domain are highly conserved among all family members; the remaining C-terminal portion shows considerable variation across members, and is specified as the hypervariable domain [11,16]. The G domain contains epitopes responsible for phosphate and guanine nucleotide ring binding. It also contains protein loops called switch region I (AA30–38) and switch region II (AA59–67) that undergo structural changes upon GTP binding and form interaction surfaces with effectors in a GTP-dependent manner. Crystal structure studies of the RASGDP-GAP complex have shown that the GTP hydrolysis step relies on the insertion of an ‘arginine finger’ by the RasGAP near the b and g phosphates of GTP resulting in neutralization of a negative charge necessary for GTP hydrolysis [19]. The common doi: 10.1586/14737159.2015.986102
oncogenic RAS mutations targeting 12G and 13G introduce amino acids with side chains that interfere with this process causing accumulation of the GTP-bound ‘on’ RAS protein. The third most common oncogenic mutation targeting Q61 in the switch II region interferes with the activation of water molecule necessary for GTP hydrolysis [20]. These mutations thus lead to accumulation of GTP-bound active RAS resulting in uncontrolled activation of downstream pathways. Phenotypically, this results in independence from extracellular growth and inhibitory signals, increased proliferation and decreased apoptosis; all of which are requirements for tumorigenesis. The hypervariable C-terminal region, while less essential for enzymatic activity, undergoes post-translational modification that is necessary for the subcellular localization of RAS proteins. All RAS proteins undergo farnelysation at the C-terminus at a CAAX motif, where C denotes a cysteine, while A stands for an aliphatic amino acid and X is any amino acid [21,22]. HRAS, NRAS and KRAS2A undergo further palmitoylation of cysteine residues at the C-terminus that anchors them to the cell membrane [23,24]. In contrast, KRAS2B lacks cysteine residues at the C-terminus and instead utilizes a polybasic stretch of lysine residues that interact with the negatively charged lipid head groups for cellular localization [25]. It has been argued that this differential localization of KRAS2B and its different microcellular environment may account, at least partly, for the overrepresentation of KRAS mutations in human cancers despite its functional similarity to the other RAS proteins [11]. Lab testing for KRAS mutation
Molecular testing of KRAS mutation is currently employed both for diagnosis (e.g., for pancreatic carcinoma using brushing sample) and for predicting response to targeted therapy (e.g., response to anti-EGFR therapies of metastatic colon cancer) [26]. The starting material for the assays includes formalinfixed paraffin-embedded (FFPE) tissue, snap frozen tissue and cytological preparations. Significant challenges in KRAS testing include limited starting material in cytology and biopsy specimens and the fragmented nature of the DNA isolated from FFPE specimens. Treatment effect from chemoradiation can also significantly reduce the amount of tumor cells. Additional challenges include the presence of heterozygosity of the mutation, the wide spectrum of mutation types, genetic heterogeneity of the tumor and the contribution of wild-type KRAS sequences from the background normal tissue [3,27]. To increase the sensitivity of the tests, often a trained pathologist selects a tumor-enriched area from histological sections for macro- or microdissection before DNA extraction. Currently, essentially all methods utilize PCR with specific primers to amplify mutation hotspots of the KRAS gene (exons 2 and 3) and then employ different downstream techniques to differentiate wildtype sequences from mutant sequences. Common techniques include methods such as Sanger sequencing, pyrosequencing, single-strand conformation polymorphism analysis (SSCP), real-time PCR with melting curve analysis and allele-specific PCR [26,27], which are briefly discussed below. Expert Rev. Mol. Diagn.
KRAS mutation testing
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Korea University on 01/09/15 For personal use only.
Sanger sequencing
Sanger sequencing of PCR-amplified DNA templates is regarded as the gold standard for KRAS testing. Sanger sequencing utilizes a mixture of deoxynucleoside triphosphates (dNTPs) and fluorescently labeled dideoxynucleoside triphosphates (ddNTPs) to generate nested fragments by chain termination during the synthesis of complimentary DNA [28]. The amplified fragments are then separated by high-resolution gel electrophoresis and the sequence is read as the fragments pass through a laser detection system. This method can provide good sequence reads of more than 500 bases, which are sufficient for KRAS mutation testing (FIGURE 2). Sanger sequencing has the advantage that it provides the entire amplified sequence and can therefore identify all possible base mutations in addition to small insertions and deletions. However, it has a low sensitivity or limit of detection of around 15–30% of mutant DNA sequence (defined as the percentage of mutant DNA to wild-type DNA) [29]. A study using automated Sanger sequencing for KRAS testing found that it had low sensitivity for a subset of codon 12 mutations in addition to an 11.1% falsepositive rate and a 6.1% false-negative rate, particularly when testing FFPE tissue samples [29]. Sanger sequencing tends to have greater utility as a confirmatory test for results obtained from other techniques that do not identify definitive specific base changes. Pyrosequencing
In pyrosequencing, DNA polymerase synthesis of complimentary DNA of a solid-support immobilized template DNA is performed with the addition of limiting amounts of specific dNTPs [28]. The inorganic pyrophosphate released after the incorporation of a complimentary base undergoes a series of enzymatic reactions ultimately resulting in a light signal that is detected as a peak in a pyrogram. In this manner, the pyrogram lists the order of incorporated complementary DNA bases and therefore the underlying DNA sequence. While pyrosequencing has been shown to have a detection limit of A) or G12V (35G>T) mutations, 15–20% are G13D mutations (38G>A) [26]. KRAS mutation status has a strong predictive value in colon cancer [47]. Investigations into the prognostic role of KRAS in colon cancer have also suggested that the G12V mutation is associated with an aggressive phenotype and may be associated with reduced overall survival [48,49]. More recent prospective randomized controlled trials have, however, failed to replicate these findings [50,51]. What is indisputable though is the predictive role of KRAS mutation status in colon cancer. The EGF signaling pathway has been increasingly targeted in cancer with the development of monoclonal antibodies and small molecule inhibitors targeting the EGFR pathway. Cetuximab and panitumumab are two anti-EGFR antibodies that have been approved by FDA as first line of treatment of metastatic colon cancer in combination with irinotecan, 5-fluorouracil and leucovorin [47]. Beginning in 2006, a series of retrospective studies were published that showed that KRAS mutations in codons 12 and 13 were negative predictors of response to anti-EGFR therapies [52–59]. Two multicenter Phase III randomized controlled trials later confirmed these findings with both trials showing better progression-free survival and overall survival with anti-EGFR therapy in patients with wild-type KRAS compared with those with mutant KRAS [57,60]. These results prompted the American Society of Clinical Oncology and the National Comprehensive Cancer Network (NCCN) to issue guidelines that recommended the KRAS mutation status (at least codons 12 and 13) be assessed prior to EGFR therapy [61–63]. Subsequently, the PRIME study demonstrated that panitumumab-FOLFOX4 was well tolerated and informahealthcare.com
Review
significantly improved progression-free survival in patients with wild-type KRAS tumors and underscores the importance of KRAS testing for patients with metastatic colorectal carcinoma [64]. The Phase III FIRE-3 Trial Data confirmed that most patients with wild-type RAS metastatic colorectal cancer benefited from first-line irinotecan, 5-fluorouracil and leucovorin plus cetuximab treatment [65]. Because of the good correlation of KRAS mutation status between the primary site and metastatic cancer, this testing can be performed on archival tissue from the original biopsy if necessary [66]. More recent studies have shown that non-exon 2 mutations are also associated with failure of response to EGFR therapy [67]. The picture has been complicated somewhat by another retrospective study that documented a positive association between G13D mutation of KRAS codon 13 and response to cetuximab, showing that the types of KRAS mutation may also be important in determining response to anti-EGFR therapy [68]. It is to be noted that KRAS mutations account for less than 40% of anti-EGFR refractory colon cancers. Mutations in BRAF, PIK3CA and NRAS and loss of PTEN expression account for a significant proportion of wild-type KRAS cases that fail to respond to anti-EGFR therapy [47]. In its updated guidelines, NCCN now recommends KRAS and NRAS testing in colon cancer. Moreover, it has been suggested by some investigators that testing for KRAS, BRAF, NRAS, PIK3CA, PTEN and MSI are necessary to identify the patients who most likely benefit from antiEGFR therapy [47]. Lung cancer
KRAS mutations are the most common somatic mutations in non-small-cell lung cancer (NSCLC), accounting for approximately 20% of all mutations with 63 documented different mutations in the COSMIC database [2]. They are strongly associated with a history of smoking, are found more commonly in adenocarcinomas and rarely in squamous cell carcinomas and are seen less commonly in Asians compared with Caucasians [69]. As in colon cancer, the majority of mutations are missense mutation involving exon 2 with the G12C mutation being more frequent. The predictive significance of KRAS mutation in NSLC is questionable since multiple studies have failed to show any association between KRAS mutations and response to conventional chemotherapy [69]. With the FDA approval of small molecule EGFR inhibitors like erlotinib and gefitinib as second- and third-line treatment of NSCLC, it is of great interest to ascertain if KRAS mutation status in NSCLC has the same predictive value for response to antiEGFR therapy as in colon cancer. Studies to date have shown that the EGFR mutation status has a higher predictive value for this purpose [70–72]. However, given that KRAS mutations are mutually exclusive to EGFR mutation and chromosomal rearrangement of ALK and ROS, the utility of KRAS mutation testing in NSCLC may lie in ruling out the other mutations in a screening algorithm. Currently, the NCCN guidelines make specific recommendations for KRAS mutation testing in NSCLC. doi: 10.1586/14737159.2015.986102
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Korea University on 01/09/15 For personal use only.
Review
Perincheri & Hui
Pancreatic & biliary cancer
KRAS mutation in other human cancers
KRAS mutations are seen in 70–90% of pancreatic ductal carcinomas and 31% of biliary tract carcinomas, respectively [73]. The majority of the mutations are again seen in exon 2, with 35G>A (G12D) being the most common [2]. KRAS mutation testing has a complimentary role in the diagnostic work-up of cytology specimens either from endoscopic retrograde cholangiopancreatography brushings or fine-needle aspiration specimens of pancreatic cystic fluid [74–76]. The presence of KRAS mutation has a high predictive value (94%) for malignancy in patients with indeterminate pancreaticobiliary cytology and should be included as an important adjuvant diagnostic marker [77]. However, molecular analysis alone has low sensitivity in the diagnosis of these lesions. KRAS, in association with cytology and loss of heterozygosity analysis, can increase both the sensitivity and positive predictive value in the diagnosis of malignant pancreatobiliary lesions [74].
In addition to the above malignancies, KRAS mutations have been documented in soft tissue, breast, esophageal, prostate and hematological malignancies among others, although the frequencies of the mutation is generally below 5% in these malignancies. KRAS mutation has been characterized in a case of imatinib-resistant chronic myelogenous leukemia [87]. KRAS mutation testing may be clinically useful in all such situations.
Thyroid carcinoma
KRAS mutations are found with varying frequencies in the various histological subtypes of thyroid carcinoma. While it is not as specific as BRAF mutation for papillary thyroid carcinoma, RAS mutations are seen in approximately 10–20% of papillary thyroid carcinomas particularly of the follicular variant, up to 50% of follicular carcinomas and have a positive predictive value of 74–88% for malignancy [78]. The mutations again tend to be missense mutations clustered in exon 2 with a few affecting Q61 [2]. However, RAS mutations are not highly specific for malignancy since they may be detectable in follicular adenomas. RAS mutations may correlate with more aggressive behavior of thyroid carcinomas [79], implying necessity for total thyroidectomy in lesions harboring them. Female genital tract tumors
KRAS mutations targeting exon 2 are seen more commonly in type 1 ovarian epithelial carcinomas including low-grade serous carcinoma and borderline tumors of the ovary. They are more often associated with an indolent course and better prognosis compared with type 2 ovarian epithelial tumors that harbor p53 mutations [80,81]. More recently, a variant allele in the KRAS-3´ untranslated region called the KRAS-variant has been described that is present in 61% of cases of hereditary breast and ovarian carcinoma syndrome [82]. This mutation alters a let-7 miRNA binding site in the KRAS-3´ untranslated region leading to deregulated levels of KRAS with low levels of background let-7 miRNA. KRAS mutations have also been detected in endometrial mucinous lesions [83,84] and have been linked to the pathogenesis to type I endometrial carcinomas that tend to be low grade with endometrioid morphology [85] and a favorable prognosis. Moreover, a recent study has shown that the nature of KRAS mutation may affect tumor behavior. In this study, tumors with KRAS amplification have a worse behavior than tumors with KRAS point mutations, again underlying the different mechanisms by which KRAS can influence tumor behavior [86]. doi: 10.1586/14737159.2015.986102
Expert commentary
KRAS mutation testing has significant clinical applications in both diagnostic and predictive scenarios. Currently, it has maximum utility in stratifying metastatic colorectal carcinoma patients for treatment with EGFR inhibitors and in diagnostic work-up of cytology specimens. KRAS testing has more of an adjunct role in the diagnostic work-up of thyroid nodules and pancreatic lesions, where the results should be interpreted in the context of histopathologic findings along with clinical and imaging data. The methodologies used in molecular diagnostic laboratories are currently dictated by the clinical specimen types and technical resources available; however, they are usually within the purview of most tertiary medical centers. Five-year view
KRAS has emerged as an essential molecular marker in the clinical practice of oncology. While molecular testing for KRAS mutation is primarily used for stratifying patients for antiEGFR therapy of colorectal and lung cancers at current time, its role is likely to expand in the near future. Numerous clinical trials exploring a plethora of small molecules for precision cancer therapies are likely to open many more venues for KRAS testing, likely in a setting of combinatorial therapeutic strategy and requiring not only characterizing KRAS mutations, but also identifying mutations of both upstream and downstream effectors. The trend is most visible already in the treatment of metastatic colorectal cancer where BRAF and NRAS testing are recommended along with KRAS. Novel technologies such as non-invasive blood-based assays to analyze circulating DNA for somatic mutations [88], digital PCR [89,90], Sequenom (a mass spectrometry genotyping assay that is suitable for broad mutation screening) [91] and the advent of nextgeneration sequencing are poised to further refine the landscape of clinical KRAS mutation analysis. Notwithstanding the challenges of working with FFPE-extracted DNA, nextgeneration sequencing can be used on archival tissue and has the potential to generate more actionable data. Next-generation sequencing is capable of increasing the clinical sensitivity of the test without decreasing the specificity of the analysis [92]. It will likely replace Sanger sequencing as the reference technique for diagnostic detection of KRAS mutation in archival tumor tissues [93]. On the other hand, tumor heterogeneity and the technical expertise needed to extract clinically important mutations from background noise will likely keep it a niche Expert Rev. Mol. Diagn.
KRAS mutation testing
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Korea University on 01/09/15 For personal use only.
expertise confined to specialized labs. A probable scenario that can be envisaged is one in which local molecular diagnostic labs perform routine PCR-based assays for screening purposes as these will, in theory, capture the vast majority of driver mutations, such as mutant KRAS. Once these are confirmed negative, the specimens may then be triaged to a reference lab for additional esoteric testing. Cancer molecular testing is a rapidly evolving and challenging field for which, in the era of precision medicine, the next decade is likely to be more exciting and rewarding.
Review
Acknowledgement
The authors thank M Talmor for her outstanding technical assistance in the preparation of this work. Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.
Key issues • Activating mutations in the RAS family GTPases that lock the RAS proteins in the GTP bound ‘on’ state are associated with 20–30% of all human malignancies with KRAS mutations accounting for a disproportionate majority. • A variety of molecular methods including Sanger sequencing, high-resolution melting analysis, single-strand conformation polymorphism analysis and allele-specific PCR methods that target mutation hotspots in KRAS exons 2 and 3 are currently employed in clinical testing of KRAS mutations and have shown good general concordance in comparative studies. • National Comprehensive Cancer Network guidelines recommend KRAS mutation testing in the clinical work-up of metastatic colorectal cancer where multiple clinical trials have shown that wild-type KRAS status is associated with a better response to anti-EGFR therapy. • Diagnostically, KRAS mutation is useful in the work-up of cytology specimens like pancreatobiliary brush specimens and thyroid fineneedle aspiration material where it can increase the positive predictive value for malignancy. • Novel methods such as next-generation sequencing and mass spectrometry genotyping assays have been shown to increase clinical sensitivity of KRAS mutation testing in clinical samples and are likely to replace current methods in the era of targeted combinatorial therapy.
References
6.
Scolnick EM, Rands E, Williams D, Parks WP. Studies on the nucleic acid sequences of Kirsten sarcoma virus: a model for formation of a mammalian RNA-containing sarcoma virus. J Virol 1973;12(3):458-63
7.
Perucho M, Goldfarb M, Shimizu K, et al. Human-tumor-derived cell lines contain common and different transforming genes. Cell 1981;27(3 Pt 2):467-76
8.
Shih C, Shilo BZ, Goldfarb MP, et al. Passage of phenotypes of chemically transformed cells via transfection of DNA and chromatin. Proc Natl Acad Sci USA 1979;76(11):5714-18
Papers of special note have been highlighted as: • of interest •• of considerable interest 1.
•
Malumbres M, Barbacid M. RAS oncogenes: the first 30 years. Nature Rev Cancer 2003;3(6):459-65 A concise summary of the major developments in understanding the biology of RAS oncogenes and their role in disease.
2.
Bamford S, Dawson E, Forbes S, et al. The COSMIC (Catalogue of Somatic Mutations in Cancer) database and website. Br J Cancer 2004;91(2):355-8
3.
Jancik S, Drabek J, Radzioch D, Hajduch M. Clinical relevance of KRAS in human cancers. J Biomed Biotechnol 2010;2010:150960
4.
Kirsten WH, Mayer LA. Morphologic responses to a murine erythroblastosis virus. J Natl Cancer Ins 1967;39(2):311-35
5.
Ellis RW, Defeo D, Shih TY, et al. The p21 src genes of Harvey and Kirsten sarcoma viruses originate from divergent members of a family of normal vertebrate genes. Nature 1981;292(5823):506-11
informahealthcare.com
9.
10.
Santos E, Martin-Zanca D, Reddy EP, et al. Malignant activation of a K-ras oncogene in lung carcinoma but not in normal tissue of the same patient. Science 1984;223(4637): 661-4 Rajalingam K, Schreck R, Rapp UR, Albert S. Ras oncogenes and their downstream targets. Biochim Biophys Acta 2007;1773(8):1177-95
11.
Ellis CA, Clark G. The importance of being K-Ras. Cell Signal 2000;12(7):425-34
12.
Barbacid M. ras genes. Ann Rev Biochem 1987;56:779-827
13.
Plowman SJ, Berry RL, Bader SA, et al. K-ras 4A and 4B are co-expressed widely in human tissues, and their ratio is altered in sporadic colorectal cancer. J Exp Clin Cancer Res 2006;25(2):259-67
14.
Johnson L, Greenbaum D, Cichowski K, et al. K-ras is an essential gene in the mouse with partial functional overlap with N-ras. Genes Dev 1997;11(19):2468-81
15.
Khalaf WF, White H, Wenning MJ, et al. K-Ras is essential for normal fetal liver erythropoiesis. Blood 2005;105(9):3538-41
16.
Schubbert S, Shannon K, Bollag G. Hyperactive Ras in developmental disorders and cancer. Nature Rev Cancer 2007;7(4): 295-308
17.
Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web. Nature Rev Cancer 2011;11(11):761-74
•
A comprehensive review of RAS oncogenes and their role in cancer.
18.
Downward J. Cancer: a tumour gene’s fatal flaws. Nature 2009;462(7269):44-5
19.
Scheffzek K, Ahmadian MR, Kabsch W, et al. The Ras-RasGAP complex: structural basis for GTPase activation and its loss in
doi: 10.1586/14737159.2015.986102
Review
Perincheri & Hui
oncogenic Ras mutants. Science 1997; 277(5324):333-8 ••
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Korea University on 01/09/15 For personal use only.
20.
21.
Outstanding review describing the structural basis of oncogenic RAS mutations. Graham DL, Eccleston JF, Lowe PN. The conserved arginine in rho-GTPase-activating protein is essential for efficient catalysis but not for complex formation with Rho.GDP and aluminum fluoride. Biochemistry 1999; 38(3):985-91 Magee AI, Newman CM, Giannakouros T, et al. Lipid modifications and function of the ras superfamily of proteins. Biochem Soc Trans 1992;20(2):497-9
22.
Cox AD, Der CJ. Protein prenylation: more than just glue? Curr Opin Cell Biol 1992; 4(6):1008-16
23.
Dudler T, Gelb MH. Palmitoylation of Ha-Ras facilitates membrane binding, activation of downstream effectors, and meiotic maturation in Xenopus oocytes. J Biol Chem 1996;271(19):11541-7
24.
25.
Milligan G, Parenti M, Magee AI. The dynamic role of palmitoylation in signal transduction. Trends Biochem Sci 1995; 20(5):181-7 Jackson JH, Li JW, Buss JE, et al. Polylysine domain of K-ras 4B protein is crucial for malignant transformation. Proc Natl Acad Sci USA 1994;91(26):12730-4
26.
Anderson SM. Laboratory methods for KRAS mutation analysis. Expert Rev Mol Diagn 2011;11(6):635-42
27.
Shackelford RE, Whitling NA, McNab P, et al. KRAS Testing: a Tool for the Implementation of Personalized Medicine. Genes Cancer 2012;3(7-8):459-66
28.
Metzker ML. Emerging technologies in DNA sequencing. Genome Res 2005; 15(12):1767-76
29.
Tsiatis AC, Norris-Kirby A, Rich RG, et al. Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications. J Mol Diagn 2010;12(4):425-32
•
Excellent article evaluating the efficacy of various methods for KRAS mutation testing in the clinical setting.
30.
Dufort S, Richard MJ, de Fraipont F. Pyrosequencing method to detect KRAS mutation in formalin-fixed and paraffin-embedded tumor tissues. Analytical Biochem 2009;391(2):166-8
31.
Ogino S, Kawasaki T, Brahmandam M, et al. Sensitive sequencing method for KRAS mutation detection by
doi: 10.1586/14737159.2015.986102
embedded tissue using mutant-enriched PCR and reverse-hybridization. J Mol Diagn 2009;11(6):508-13
Pyrosequencing. J Mol Diagn 2005;7(3): 413-21 32.
Weichert W, Schewe C, Lehmann A, et al. KRAS genotyping of paraffin-embedded colorectal cancer tissue in routine diagnostics: comparison of methods and impact of histology. J Mol Diagn 2010; 12(1):35-42
44.
Whitehall V, Tran K, Umapathy A, et al. A multicenter blinded study to evaluate KRAS mutation testing methodologies in the clinical setting. J Mol Diagn 2009; 11(6):543-52
33.
Gao J, Li YY, Sun PN, Shen L. Comparative analysis of dideoxy sequencing, the KRAS StripAssay and pyrosequencing for detection of KRAS mutation. World J Gastroenterol 2010;16(38):4858-64
45.
Forrester K, Almoguera C, Han K, et al. Detection of high incidence of K-ras oncogenes during human colon tumorigenesis. Nature 1987;327(6120): 298-303
34.
Krypuy M, Newnham GM, Thomas DM, et al. High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer. BMC Cancer 2006;6:295
46.
Vogelstein B, Fearon ER, Hamilton SR, et al. Genetic alterations during colorectal-tumor development. N Engl J Med 1988;319(9):525-32
47.
Tan C, Du X. KRAS mutation testing in metastatic colorectal cancer. World J Gastroenterol 2012;18(37):5171-80
48.
Andreyev HJ, Norman AR, Cunningham D, et al. Kirsten ras mutations in patients with colorectal cancer: the multicenter "RASCAL" study. J Natl Cancer Inst 1998;90(9):675-84
49.
Andreyev HJ, Norman AR, Cunningham D, et al. Kirsten ras mutations in patients with colorectal cancer: the ’RASCAL II’ study. Br J Cancer 2001;85(5):692-6
50.
Roth AD, Tejpar S, Delorenzi M, et al. Prognostic role of KRAS and BRAF in stage II and III resected colon cancer: results of the translational study on the PETACC-3, EORTC 40993, SAKK 60-00 trial. J Clin Oncol 2010;28(3):466-74
51.
Ogino S, Meyerhardt JA, Irahara N, et al. KRAS mutation in stage III colon cancer and clinical outcome following intergroup trial CALGB 89803. Clin Cancer Res 2009; 15(23):7322-9
52.
Lievre A, Bachet JB, Le Corre D, et al. KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer Res 2006;66(8):3992-5
•
First published report of the correlation between KRAS mutation status and response to anti-EGFR therapy.
53.
Khambata-Ford S, Garrett CR, Meropol NJ, et al. Expression of epiregulin and amphiregulin and K-ras mutation status predict disease control in metastatic colorectal cancer patients treated with cetuximab. J Clin Oncol 2007;25(22): 3230-7
54.
Di Fiore F, Blanchard F, Charbonnier F, et al. Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by Cetuximab plus chemotherapy. Br J Cancer 2007;96(8):1166-9
35.
Franklin WA, Haney J, Sugita M, et al. KRAS mutation: comparison of testing methods and tissue sampling techniques in colon cancer. J Mol Diagn 2010;12(1): 43-50
36.
Ziai J, Hui P. BRAF mutation testing in clinical practice. Expert Rev Mol Diagn 2012;12(2):127-38
37.
Emanuel JR, Damico C, Ahn S, et al. Highly sensitive nonradioactive single-strand conformational polymorphism. Detection of Ki-ras mutations. Diagn Mol Pathol B 1996;5(4):260-4
38.
39.
40.
41.
42.
43.
Dillon DA, Johnson CC, Topazian MD, et al. The utility of Ki-ras mutation analysis in the cytologic diagnosis of pancreatobiliary neoplasma. Cancer J 2000;6(5):294-301 Hayes VM, Westra JL, Verlind E, et al. New comprehensive denaturing-gradient-gelelectrophoresis assay for KRAS mutation detection applied to paraffin-embedded tumours. Genes Chromosomes Cancer 2000;29(4):309-14 Oliner K, Juan T, Suggs S, et al. A comparability study of 5 commercial KRAS tests. Diagnostic Pathol 2010;5:23 Pritchard CC, Akagi L, Reddy PL, et al. COLD-PCR enhanced melting curve analysis improves diagnostic accuracy for KRAS mutations in colorectal carcinoma. BMC Clin Pathol 2010;10:6 Kriegshauser G, Auner V, Schuster E, et al. KRAS mutation analysis in genomic DNA isolated from formalin-fixed paraffinembedded ovarian tissue: evaluation of a strip-based reverse-hybridisation assay. J Clin Pathol 2011;64(3):252-6 Ausch C, Buxhofer-Ausch V, Oberkanins C, et al. Sensitive detection of KRAS mutations in archived formalin-fixed paraffin-
Expert Rev. Mol. Diagn.
KRAS mutation testing
55.
56.
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Korea University on 01/09/15 For personal use only.
57.
58.
59.
60.
61.
De Roock W, Piessevaux H, De Schutter J, et al. KRAS wild-type state predicts survival and is associated to early radiological response in metastatic colorectal cancer treated with cetuximab. Ann Oncol 2008; 19(3):508-15 Bibeau F, Frugier H, Boissiere-Michot F. [Responses to targeted therapies: colorectal cancer]. Ann Pathol 2009;29(Spec No 1): S74-6
PRIME study. J Clin Oncol 2010;28(31): 4697-705 65.
66.
Amado RG, Wolf M, Peeters M, et al. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol 2008;26(10):1626-34 Freeman DJ, Juan T, Reiner M, et al. Association of K-ras mutational status and clinical outcomes in patients with metastatic colorectal cancer receiving panitumumab alone. Clin Colorectal Cancer 2008;7(3): 184-90 Adelstein BA, Dobbins TA, Harris CA, et al. A systematic review and meta-analysis of KRAS status as the determinant of response to anti-EGFR antibodies and the impact of partner chemotherapy in metastatic colorectal cancer. Eur J Cancer 2011;47(9):1343-54 Karapetis CS, Khambata-Ford S, Jonker DJ, et al. K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med 2008;359(17):1757-65 Allegra CJ, Jessup JM, Somerfield MR, et al. American Society of Clinical Oncology provisional clinical opinion: testing for KRAS gene mutations in patients with metastatic colorectal carcinoma to predict response to anti-epidermal growth factor receptor monoclonal antibody therapy. J Clin Oncol 2009;27(12):2091-6
••
Excellent article summarizing the evidence behind recommendations for KRAS testing in metastatic colon cancer.
62.
Engstrom PF, Arnoletti JP, Benson AB 3rd, et al. NCCN Clinical Practice Guidelines in Oncology: colon cancer. J Natl Compr Canc Netw 2009;7(8):778-831
63.
Engstrom PF, Arnoletti JP, Benson AB 3rd, et al. NCCN Clinical Practice Guidelines in Oncology: rectal cancer. J Natl Compr Canc Netw 2009;7(8):838-81
64.
Douillard JY, Siena S, Cassidy J, et al. Randomized, phase III trial of panitumumab with infusional fluorouracil, leucovorin, and oxaliplatin (FOLFOX4) versus FOLFOX4 alone as first-line treatment in patients with previously untreated metastatic colorectal cancer: the
informahealthcare.com
Heinemann V, von Weikersthal LF, Decker T, et al. FOLFIRI plus cetuximab versus FOLFIRI plus bevacizumab as first-line treatment for patients with metastatic colorectal cancer (FIRE-3): a randomised, open-label, phase 3 trial. Lancet Oncol 2014;15(10):1065-75 Etienne-Grimaldi MC, Formento JL, Francoual M, et al. K-Ras mutations and treatment outcome in colorectal cancer patients receiving exclusive fluoropyrimidine therapy. Clin Cancer Res 2008;14(15): 4830-5
67.
Douillard JY, Oliner KS, Siena S, et al. Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer. N Engl J Med 2013;369(11):1023-34
68.
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(16):1812-20
69.
Roberts PJ, Stinchcombe TE. KRAS mutation: should we test for it, and does it matter? J Clin Oncol 2013;31(8):1112-21
70.
Zhu CQ, da Cunha Santos G, Ding K, et al. Role of KRAS and EGFR as biomarkers of response to erlotinib in National Cancer Institute of Canada Clinical Trials Group Study BR.21. J Clin Oncol 2008;26(26):4268-75
71.
Brugger W, Triller N, Blasinska-Morawiec M, et al. Prospective molecular marker analyses of EGFR and KRAS from a randomized, placebo-controlled study of erlotinib maintenance therapy in advanced non-small-cell lung cancer. J Clin Oncol 2011;29(31):4113-20
72.
Hirsch FR. K-ras mutations in lung cancer: the "mysterious mutation". Clin Lung Cancer 2006;8(1):11-12
73.
Miglio U, Oldani A, Mezzapelle R, et al. KRAS mutational analysis in ductal adenocarcinoma of the pancreas and its clinical significance. Pathol Res Pract 2014; 210(5):307-11
74.
Somoza AD, Aly FZ. Utility of molecular tests in cytopathology. CytoJournal 2014;11:5
75.
Kipp BR, Fritcher EG, Clayton AC, et al. Comparison of KRAS mutation analysis and FISH for detecting pancreatobiliary tract cancer in cytology specimens collected during endoscopic retrograde
Review
cholangiopancreatography. J Mol Diagn 2010;12(6):780-6 76.
Sturm PD, Rauws EA, Hruban RH, et al. Clinical value of K-ras codon 12 analysis and endobiliary brush cytology for the diagnosis of malignant extrahepatic bile duct stenosis. Clin Cancer Res 1999;5(3):629-35
77.
Cai G, Mahooti S, Lipata FM, et al. Diagnostic value of K-ras mutation analysis for pancreaticobiliary cytology specimens with indeterminate diagnosis. Cancer Cytopathol 2012;120(5):313-18
78.
Nikiforov YE. Molecular diagnostics of thyroid tumors. Arch Pathol Lab Med 2011;135(5):569-77
79.
Garcia-Rostan G, Zhao H, Camp RL, et al. ras mutations are associated with aggressive tumor phenotypes and poor prognosis in thyroid cancer. J Clin Oncol 2003;21(17): 3226-35
80.
Shih Ie M, Kurman RJ. Ovarian tumorigenesis: a proposed model based on morphological and molecular genetic analysis. Am J Pathol 2004;164(5):1511-18
81.
Singer G, Oldt R 3rd, Cohen Y, et al. Mutations in BRAF and KRAS characterize the development of low-grade ovarian serous carcinoma. J Natl Cancer Inst 2003;95(6): 484-6
82.
Ratner E, Lu L, Boeke M, et al. A KRAS-variant in ovarian cancer acts as a genetic marker of cancer risk. Cancer Res 2010;70(16):6509-15
83.
Alomari A, Abi-Raad R, Buza N, Hui P. Frequent KRAS mutation in complex mucinous epithelial lesions of the endometrium. Mod Pathol 2014;27(5): 675-80
84.
Ip PP, Irving JA, McCluggage WG, et al. Papillary proliferation of the endometrium: a clinicopathologic study of 59 cases of simple and complex papillae without cytologic atypia. Am J Surg Pathol 2013; 37(2):167-77
85.
Xiong J, He M, Jackson C, et al. Endometrial carcinomas with significant mucinous differentiation associated with higher frequency of k-ras mutations: a morphologic and molecular correlation study. Int J Gynecol Cancer 2013;23(7): 1231-6
86.
Birkeland E, Wik E, Mjos S, et al. KRAS gene amplification and overexpression but not mutation associates with aggressive and metastatic endometrial cancer. Br J Cancer 2012;107(12):1997-2004
87.
Agarwal A, Eide CA, Harlow A, et al. An activating KRAS mutation in
doi: 10.1586/14737159.2015.986102
Review
Perincheri & Hui
adenocarcinoma. Cancer Biol Ther 2012; 13(10):871-9
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Korea University on 01/09/15 For personal use only.
imatinib-resistant chronic myeloid leukemia. Leukemia 2008;22(12):2269-72 88.
Holdhoff M, Schmidt K, Donehower R, Diaz LA Jr. Analysis of circulating tumor DNA to confirm somatic KRAS mutations. J Natl Cancer Inst 2009;101(18):1284-5
89.
Azuara D, Ginesta MM, Gausachs M, et al. Nanofluidic digital PCR for KRAS mutation detection and quantification in gastrointestinal cancer. Clin Chem 2012; 58(9):1332-41
90.
Zhou B, Irwanto A, Guo YM, et al. Exome sequencing and digital PCR analyses reveal novel mutated genes related to the metastasis of pancreatic ductal
doi: 10.1586/14737159.2015.986102
91.
92.
Arcila M, Lau C, Nafa K, Ladanyi M. Detection of KRAS and BRAF mutations in colorectal carcinoma roles for high-sensitivity locked nucleic acid-PCR sequencing and broad-spectrum mass spectrometry genotyping. J Mol Diagn 2011;13(1):64-73 de Biase D, Visani M, Baccarini P, et al. Next generation sequencing improves the accuracy of KRAS mutation analysis in endoscopic ultrasound fine needle aspiration pancreatic lesions. PLoS One 2014;9(2): e87651
93.
Altimari A, de Biase D, De Maglio G, et al. 454 next generation-sequencing outperforms allele-specific PCR, Sanger sequencing, and pyrosequencing for routine KRAS mutation analysis of formalin-fixed, paraffin-embedded samples. OncoTargets Ther 2013;6:1057-64
••
Excellent review comparing next-generation sequencing with traditional methods of KRAS mutation testing in clinical specimens.
Expert Rev. Mol. Diagn.
