Mutation testing has become an essential determinant in clinical practice in decision of treatment options for patients with non-small-cell lung carcinomas. Unfortunately NSCLC tumours, in which the molecular diagnostics is carried out, are highly heterogeneous and the cytological and histological material is often insufficient to complete the analysis (small percentage of cancer cells or DNA fragmentation in the process of paraffin embedding). Direct sequencing is still a frequently used method despite having low sensitivity and being time-consuming and labour-intensive. However, direct sequencing and particularly next generation sequencing (technology based on reversible dye terminators, sequencing by ligation and pirosequencing) are the methods of high-throughput screening for unknown mutations. Microarrays containing oligonucleotide mutation probes are emerging as useful platforms for the diagnosis of multiple genetic abnormalities in cancer cells [17, 18].
The multiplex SNaPshot PCR (minisequencing) technique is a PCR (polymerase chain reaction)-based assay for detection of known mutations. Specific primer which anneals immediately adjacent to the mutated region is extended by one base using a fluorescently labeled ddNTPs, which are detected in capillary electrophoresis. No further extension is possible because of the ddNTP binding. This kind of reaction is being used more and more frequently because of its fast and sensitive detection of many known mutations in a single assay .
Recent advances in molecular techniques have enabled the design of sensitive detection assays based on quantitative real-time PCR, but usually with limited degree of mutation coverage. Allele-specific PCR (ASP-PCR), amplification refractory mutation system PCR (ARMS-PCR), clamp PCR and mutant-enriched PCR (ME-PCR) are among these techniques. The most frequently used is the ARMS-PCR method that can detect a known SNP (single nucleotide polymorphism). It consists of two complementary reactions: one containing an ARMS primer specific for the normal DNA sequence that cannot amplify mutant DNA at a given locus and the other one containing a mutant-specific primer that cannot amplify normal DNA. High resolution melting (HRM) real-time PCR is also a technique that might allow fast screening for mutations. The real-time PCR technology itself is highly flexible and many alternative instruments and fluorescent probe systems have been developed recently [17, 18].
For detecting polysomy, gene amplifications and the presence of fusion genes molecular probes labelled with different fluorochromes and fluorescence in situ hybridisation (FISH) technique are being used. Techniques related to FISH, but allowing to label only one gene fragment, are silver in situ hybridisation (SISH) and chromogenic in situ hybridisation (CISH). The FISH technique requires an assessment of signal quantity from labelled genes and chromosome fragments with fluorescence microscopy whereas SISH or CISH staining can be analysed in light microscope [17, 18].
Routine genetic testing for somatic mutations in lung cancer biopsies is becoming the standard for providing optimal patients care. However, it is unclear whether this testing should be routine for all lung cancer patients, because the prevalence of the most common mutations is very low especially in heavy smokers with squamous cell carcinoma. Moreover, great number of molecular biology methods and variety of biological material acquired from patients create a critical need for robust, well-validated diagnostic tests and equipment that are both sensitive and specific for mutations. An In Vitro Diagnostic Medical Device (IVD) is defined in Directive 98/79/EC of European Parliament and of the Council. IVD is described as any medical device which is a reagent, calibrator, control material, kit, equipment or system, whether used alone or in combination, intended by the manufacturer to be used in vitro for the examination of specimens, including blood and tissue donations, derived from the human body for the purpose of providing information concerning pathological state and congenital abnormalities of patients as well as to monitor therapeutic effect. IVD equipment is labelled by CE marking according to European Product Safety Regulations .
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