Definition of RAS
The RAS gene family is widely expressed in mammalian cells where encodes four small (21 kDa), cytoplasmic proteins with GTPase activity: H-Ras, K-Ras4a, K-Ras4b, and N-Ras.
They function as molecular switches transducing extracellular stimuli such as mitogens and differentiation factors to transcription factors and cell cycle proteins in the nucleus in order to promote cell growth, differentiation, proliferation and survival.
Extracellular stimuli activate firstly transmembrane tyrosine kinase receptors (RTKs) which recruits adaptor proteins that catalyse the hydrolysis of GDP to GTP on Ras. Once activated, Ras recruits and stimulates number of effectors of complex signalling network pathways including Raf/MEK/ERK mitogen activated protein kinase (MAPK) pathway, the phosphoinositide 3-kinase PI3K/Akt and Ral-GEF proteins.1
Activating mutations in members of RAS family have been found in 20–25% of human cancers. Mutations in RAS are single nucleotide point mutations that more frequently interest the exon 2 codons 12 13 and exon 3 codon 61. These mutations set proteins in a permanently activated state (GTP-bound conformation) impairing the ATPase activity.
Deregulated Ras signalling results in increased proliferation, angiogenesis, and motility, as well as in decreased apoptosis and in altered cellular metabolism.
KRAS is the most commonly mutated isoform in almost 22% of all human cancers, followed by NRAS (8%) and HRAS (3.3%).
Different types of cancer appear to be related to a particular RAS isoform mutation. KRAS mutations are most commonly found in cancers of the pancreas, colon, lung and biliary tract while NRAS mutations are more common in malignant melanoma and haematopoietic system. Lastly HRAS mutations are most common in cancers of the head and neck and urinary tract.1,2
RAS Mutations in Colorectal Cancer
In colorectal cancer KRAS is mutated in approximately 40% of cases mostly in exon 2 codons 12 (70-80%) and 13 (15-20%). The remaining mutations are mainly located in exon 3 codons 59-61 and in exon 4, which includes codons 117 and 146. Mutations in NRAS are present in approximately 3% to 5% of colorectal cancer samples particularly in exon 3 codon 61 (60%) and in exon 2 codons 12, 13.
NRAS mutations are typically mutually exclusive with KRAS mutations. HRAS mutations represent a negligible event.3
Mutations in KRAS are considered an early event in colorectal carcinogenesis and are maintained during the colorectal cancer development, as demonstrated by the highly concordant rate (almost 95%) in paired primary cancers and metastatic samples, except between primary tumours and metastatic lymph nodes.4 Mutations in KRAS or NRAS lead to continuous activation MAPK pathway even if the EGFR is inactivated by drugs.5
RAS as a Prognostic Biomarker in Colorectal Cancer
The prognostic role of RAS mutations in patients with colorectal cancer remains controversial.
A negative effect of KRAS mutations have been reported both in the adjuvant and in metastatic setting but no definitive conclusions have been drawn.6
In a retrospective series of chemo-refractory patients with metastatic colorectal cancer treated with anti-EGFR monoclonal antibodies a poorer median overall survival has been observed in NRAS and KRAS mutated tumours compared to all wild type ones.7
Some authors described a possible negative prognostic role for RAS mutations in patients undergoing liver surgery for colorectal cancer metastases, however this data has not been confirmed in a recent series.8
RAS as a Predictive Biomarker in Colorectal Cancer
Several clinical trials highlighted the role KRAS exon 2 (codons 12 and 13) mutations as biomarkers of resistance to anti-EGFR monoclonal antibodies cetuximab and panitumumab.9 De Roock et al. reported longer progression-free survival and overall survival in metastatic colorectal cancer patients carrying a KRAS mutation in codon 13 compared with patients harbouring other KRAS mutations when treated with cetuximab, however this data has not yet been confirmed in prospective clinical trial.10 Recently retrospective and prospective trials have demonstrated the inefficacy of anti-EGFR monoclonal antibodies in metastatic colorectal cancer harbouring either mutations both in KRAS exons 3-4 and in NRAS exons 2-3-4. Sorich and colleagues published a meta-analysis of several randomised controlled trials evaluating EGFR antibodies in different lines of therapy for metastatic colorectal cancer. They found firstly that roughly 20% of KRAS exon 2 wild-type tumours harboured other RAS mutation that impair the effectiveness of anti-EGFR antibodies and secondly confirmed that anti-EGFR antibodies treatment was superior for all–RAS wild-type tumours compared with the expanded RAS mutant subgroup in terms of both progression-free survival and overall survival. Interestingly efficacy was not significantly different between the other RAS mutations and KRAS exon 2–mutant subgroups.11
For this reason the European Medicines Agency has recently updated prescribing indications for panitumumab and cetuximab, restricting their use to the 47% of patients with RAS wild type metastatic colorectal cancer.12,13
RAS Testing Recommendations in Colorectal Cancer
RAS mutation status should be obtained by a validated test carried out in an accredited (certified) institution that includes appropriate quality controls.
Different methods can be used to detect RAS mutations in colorectal cancer specimens, such as mutation-specific real-time polymerase chain reaction (RT-PCR), Sanger sequencing, pyrosequencing, BEAMing technique, next-generation sequencing and dideoxy nucleotide sequencing. There are several manufacturers of targeted genetic tests that can detect RAS mutations in colorectal cancer samples. Tissue materials of either primary or metastatic lesions are applicable for RAS mutation testing since the concordance rate of the mutation status between primary tumours and metastatic sites reached 93%.14
Which Technique and Which Algorithm Should be Used for the Analysis of the RAS Status in Colorectal Cancer?
All methods used to detect RAS mutations have advantages and disadvantages, and the choice to use one over the other is usually based on current local practices and experience in the different clinical laboratories. Parameters that should be considered when choosing a suitable companion diagnostic test include sensitivity, specificity, limit of analytical sensitivity and failure rates.
Methods to increase the sensitivity and specificity and to reduce the failure rates include:
- validating the chosen method thoroughly through comparison with 'gold-standard' methods,
- performing macrodissection of specimens to increase the sensitivity of the technique,
- choosing small amplicons for PCR amplification to reduce the failure rate due to DNA degradation,
- ongoing validation of the method through application of best practice and participation in external quality controls.14
In line with the clinical practice guidelines of the European Society for Medical Oncology, patients with metastatic colorectal cancer must be screened for RAS mutations before initiating any anti-EGFR therapy since it has been widely demonstrated the ineffectiveness of these treatments in metastatic colorectal cancer harbouring any RAS mutation; it also would avoid drug-induced toxicity and unnecessary cost expenses.15
- Prior IA, Lewis PD, Mattos C. A comprehensive survey of Ras mutations in cancer. Cancer Res 2012;72:2457–2467.
- Forbes SA, Bindal N, Bamford S, et al. COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic Acids Res 2011;39:D945–50.
- JL Bos, ER Fearon, SR Hamilton, et al. Prevalence of ras gene mutations in human colorectal cancers. Nature 1987;327:293–297.
- Han CB, Li F, Ma JT, Zou HW. Concordant KRAS mutations in primary and metastatic colorectal cancer tissue specimens: a meta-analysis and systematic review. Cancer Invest 2012;30:741–747.
- S Benvenuti, A Sartore-Bianchi, F Di Nicolantonio, et al. Oncogenic activation of the RAS/RAF signaling pathway impairs the response of metastatic colorectal cancers to anti-epidermal growth factor receptor antibody therapies, Cancer Res 2007;67:2643–2648.
- T Yokota. “Are KRAS/BRAF Mutations Potent Prognostic And/or Predictive Biomarkers in Colorectal Cancers?” Anti-Cancer Agents in Medicinal Chemistry 2012;12(2):163–171.
- Schirripa M, Cremolini C, Loupakis F, et al. Role of NRAS mutations as prognostic and predictive markers in metastatic colorectal cancer. Int J Cancer 2015;136: 83–90.
- Schirripa M, Bergamo F, Cremolini C, et al. BRAF and RAS mutations as prognostic factors in metastatic colorectal cancer patients undergoing liver resection. British Journal of Cancer 2015;112:1921–1928.
- Vecchione L, Jacobs B, Normanno N, et al. EGFR-targeted therapy. Exp Cell Res 2011;317(19):2765-71.
- 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):1812–1820.
- Sorich MJ, Wiese MD, Rowland A, et al. Extended RAS mutations and anti-EGFR monoclonal antibody survival benefit in metastatic colorectal cancer: a meta-analysis of randomized, controlled trials. Ann Oncol 2015;26(1):13-21.
- European Medicines Agency, Committee for Medicinal Products for Human Use. Erbitux Assessment report EMA/CHMP/701107/2013.
- European Medicines Agency, Committee for Medicinal Products for Human Use. Vectibix Assessment report EMA/413562/2013.
- Wong N, Gonzalez D, Salto-Tellez M, et al. RAS testing of colorectal carcinoma – a guidance document from the association of clinical pathologists molecular pathology and diagnostics group. J Clin Pathol 2014;67: 751–757.
- Van Cutsem E, Cervantes A, Nordlinger B, et al. Metastatic colorectal cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol (2014) 25 (suppl 3): iii1-iii9.