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KRAS mutation analysis is currently used as a predictive marker of therapy response in the setting of anti EGFR therapy in metastatic colorectal carcinoma and pulmonary adenocarcinoma. Anti EGFR therapy is currently limited to tumors lacking molecular diagnostic evidence for KRAS or BRAF mutations.
Background and Rationale:
KRAS (Kirsten rat sarcoma virus) gene, mapped to 12p12.1, is a member of the RAS gene family that also includes HRAS and NRAS. RAS genes encode GDP/GTP-binding proteins involved in signal transduction during cellular proliferation, differentiation and senescence.
Activation of KRAS, its upstream regulators (eg. EGFR) or downstream pathway members (eg. RAF/MAP) are common events during oncogenesis. Several members of the KRAS signal transduction pathway are attractive targets of therapy in a variety of solid tumors1-3. It is in the setting of targeted therapy using tyrosine kinase inhibitors that KRAS mutational status has acquired a role as a predictive marker of therapeutic response.
It is estimated that 17 to 25% of all human tumors harbor an activating KRAS mutation4. Although pancreatic carcinomas carry the highest frequency of KRAS mutations (over 90%), KRAS mutations are also frequently encountered in pulmonary non-small cell and colorectal carcinomas (approximately 40% each). Critical regions of oncogenic KRAS activating mutations include codons 12, 13, 59, 61, and 635-7. Such mutations cause KRAS protein to accumulate in its active GTP-bound state8 leading to constitutive activation of downstream signaling. Such activating mutations provide a mechanism to bypass the antitumor effect of therapeutic strategies directed to upstream receptor tyrosine kinase regulators such as EGFR. Indeed, several clinical trials have now demonstrated lack of efficacy of anti EGFR agents such as Erlotinib (small molecule agent) and Cetuximab or Panitumumab (anti EGFR monoclonal antibodies) in colorectal and non-small cell lung carcinoma patients 5, 6, 9-13.
In colorectal cancers, somatic KRAS mutations predominantly involve codons 12 and 13 of exon 1. Among the seven most commonly encountered mutations, six involve codon 12 and one mutation affects codon 13. The next most frequent mutation is in codon 61; however, most assays currently in use do not target this mutation. Geographical variations in the incidence of the different mutations are reported and are thought to be potentially attributed to geographic variations in carcinogen exposure.
In pulmonary adenocarcinoma, somatic KRAS mutations are far more frequent in tumors that are associated with a history of cigarette smoking14. The G12C mutation in codon 12 is by far the most frequent mutation in these tumors.
Although primary tumor samples were used in the original clinical trials that unveiled the predictive role of KRAS mutational analysis, both metastatic and primary tumor specimens are currently used in clinical practice for mutational analysis 15. Several studies have indicated a reliable correlation of KRAS mutation status in primary and metastatic tumor specimens16, 17.
Several molecular methodology platforms are currently in use for the detection of KRAS mutations in formalin fixed paraffin embedded (FFPE) tumor samples (table). Usually, manual microdissection is necessary to increase the fraction of tumor cells in the sample. Intratumoral heterogeneity, the presence of wild type alleles in the tumor cells, and dilution by stromal and inflammatory cells pose additional detection and sensitivity challenges. Therefore, a highly sensitive method is required for the detection of KRAS mutations. Methods currently in use are sensitive to about 10% mutant alleles in a sample, or tissue with at least 20% tumor containing a mutation of KRAS in one allele.
Allele specific amplification and detection systems include the amplification refractory mutation system (ARMS)/ real-time PCR detection method,18 single nucleotide extension, the oligonucleotide ligation assay, and short oligonucleotide mass analysis (SOMA). These methods will only detect the specific mutations covered by the assay. Additional mutations can be detected by mutation screening methods, such as high resolution melting analysis19, single stranded conformation polymorphism (SSCP) analysis, and restriction fragment length polymorphism (RFLP) analysis.
Sequencing techniques can detect all possible mutations in the targeted region. Traditional Sanger sequencing is not in general use for KRAS mutation detection due to its low sensitivity for minor alleles in a heterogeneous mixture of tumor and inflammatory / stromal tissue. Pyrosequencing is as sensitive as allele specific amplification methods.
Hybridization-based detection systems, including chip-based and reverse dot-blot strip assays, permit simultaneous screening for multiple mutations in one assay.
There is increasing interest in non-invasive techniques to detect KRAS mutations. Among these methods is magnetic capture and separation of tumor cells in peripheral blood, followed by nested allele-specific amplification of KRAS. The clinical utility of such alternate methods is yet to be proven.
|Sanger sequencing||All possible mutations may be detected in target region||Low sensitivity for minor alleles (20%); labor intensive|
|Pyrosequencing20||High sensitivity; can customize assay design||Requires specialized equipment|
|Allele-specific real time PCR (ARMS/ Scorpions)18||High sensitivity; kit based||Expensive; limited to 7 common mutations|
|Single nucleotide extension||High sensitivity; can customize assay design||Multiplexing necessary|
|Allele specific PCR and high resolution melting analysis21||High sensitivity; can customize assay design||May detect minority populations (?mutations not biologically relevant)|
|Short oligonucleotide mass analysis (SOMA)22||High sensitivity; can customize assay design||May detect minority populations (?mutations not biologically relevant)May detect minority populations (?mutations not biologically relevant)|
|Chip based assays||Ease of multiplexing||Requires specialized equipment|
|Strip Assay||Kit based||Limited to 10 common mutations|
|1.||Downward J. Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer. 2003;3:11-22.|
|2.||Tsao MS, Sakurada A, Cutz JC et al. Erlotinib in lung cancer - molecular and clinical predictors of outcome. N Engl J Med. 2005;353:133-44.|
|3.||Cunningham D, Humblet Y, Siena S et al. Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J Med. 2004;351:337-45.|
|4.||Kranenburg O. The KRAS oncogene: past, present, and future. Biochim Biophys Acta. 2005;1756:81-2.|
|5.||Lievre A, Bachet JB, Boige V et al. KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab. J Clin Oncol. 2008;26:374-9.|
|6.||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;66:3992-5.|
|7.||Toyooka S, Tsukuda K, Ouchida M et al. Detection of codon 61 point mutations of the K-ras gene in lung and colorectal cancers by enriched PCR. Oncol Rep. 2003;10:1455-9.|
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|10.||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;96:1166-9.|
|11.||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;19:508-15.|
|12.||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;26:1626-34.|
|13.||Eberhard DA, Johnson BE, Amler LC et al. Mutations in the epidermal growth factor receptor and in KRAS are predictive and prognostic indicators in patients with non-small-cell lung cancer treated with chemotherapy alone and in combination with erlotinib. J Clin Oncol. 2005;23:5900-9.|
|14.||Ahrendt SA, Decker PA, Alawi EA et al. Cigarette smoking is strongly associated with mutation of the K-ras gene in patients with primary adenocarcinoma of the lung. Cancer. 2001;92:1525-30.|
|15.||Gattenlohner S, Germer C, Muller-Hermelink HK. K-ras mutations and cetuximab in colorectal cancer. N Engl J Med. 2009;360:835; author reply 835-6.|
|16.||Santini D, Loupakis F, Vincenzi B et al. High concordance of KRAS status between primary colorectal tumors and related metastatic sites: implications for clinical practice. Oncologist. 2008;13:1270-5.|
|17.||Artale S, Sartore-Bianchi A, Veronese SM et al. Mutations of KRAS and BRAF in primary and matched metastatic sites of colorectal cancer. J Clin Oncol. 2008;26:4217-9.|
|18.||Newton CR, Graham A, Heptinstall LE et al. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res. 1989;17:2503-16.|
|19.||Do H, Krypuy M, Mitchell PL, Fox SB, Dobrovic A. High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies. BMC Cancer. 2008;8:142.|
|20.||Ogino S, Kawasaki T, Brahmandam M et al. Sensitive sequencing method for KRAS mutation detection by Pyrosequencing. J Mol Diagn. 2005;7:413-21.|
|21.||Krypuy M, Newnham GM, Thomas DM, Conron M, Dobrovic A. 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.|
|22.||Lleonart ME, Ramon y Cajal S, Groopman JD, Friesen MD. Sensitive and specific detection of K-ras mutations in colon tumors by short oligonucleotide mass analysis. Nucleic Acids Res. 2004;32:e53.|