The National Institute for Health and Care Excellence (NICE) is a UK based organisation that works to create universal comprehensive guidelines and regulations for many aspects of Medicine and Science, with an aim to maximise patient safety and therapeutic effectiveness. Whilst it primarily provides guidance for UK government bodies, the National Health Service and UK Pharmaceutical companies, it also has a large influence internationally. In the UK a majority of healthcare practices design their best practice guidelines to NICE regulations (https://www.nice.org.uk/).
NICE has produced comprehensive guidelines for treatment of lung cancer (described earlier) and also for the use of EGFR mutation detection in NSCLC. Clinical trials have shown that lung cancer patients who are positive for EGFR activation mutations respond better to tyrosine kinase inhibitor therapy compared to treatment with standard chemotherapy (Hagiwara & Kobayashi, 2013). Therefore it is logical that all patients with NSCLC be tested for EGFR mutations (key pathogenic mutations described in section 1.3.1), as this will have a
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significant influence on clinical management choices made by their attending physician, ultimately leading to an improved outcome from more appropriate therapy.
The procedure for testing for EGFR mutations in NSCLC in UK diagnostic laboratories is not standardised, and there is some variation in the methods used. The methods for testing for EGFR mutations can be divided into two key types: targeted mutant detection and mutation screening. With targeted methods, only known mutations are analysed, whereas with mutation screening all known and novel variants are screened for. Many laboratories use both of these types of method for EGFR mutant detection. NICE has evaluated many tests currently in diagnostic use in NHS laboratories. Each is summarised in sections 1.4.1 – 1.4.8.
1.4.1 Qiagen™ Therascreen EGFR RGQ PCR Kit
This is a real-time PCR assay that detects 29 EGFR mutations (for full list, see section 2.2.6.1). The test first requires DNA to be extracted from FFPE samples using the QIAamp DNA FFPE Tissue Kit, and then a control assay needs to be performed to quantify the total extracted DNA. The Therascreen PCR can then be performed to detect EGFR mutants in the sample. To detect the mutant, the Therascreen PCR uses two technologies: Amplification Refractory Mutation System (ARMS) for specific amplification of the mutants; and Scorpions for detection of the amplified regions. For diagrams of the Scorpion-ARMS technology, see section 2.1.
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1.4.2 Roche Molecular Systems Cobas® EGFR Mutation Testing Kit
This is another real-time PCR assay for EGFR mutations, able to detect 41 different variants. Like Therascreen, the DNA needs to be extracted from the clinical sample using a specific kit, the Cobas® DNA Sample Preparation Kit. The extracted DNA can then be analysed using the Cobas® EGFR assay. The PCR uses complimentary primer pairs and fluorescently labelled probes to detect the mutations. The assay is run using the Cobas® z480 analyser which automates amplification and detection, and the software provides the laboratory with automated test result reporting.
1.4.3 Sanger Sequencing
This method of analysis allows all mutation types to be detected, both known and novel. Sanger sequencing is a very commonly used technique for disease mutant detection, however there is considerable variation in the way the procedures are performed. Typically, DNA will be extracted from the clinical sample and PCR used to amplify the region of interest, in this case, various regions of exons 19-21 of the EGFR gene. The amplified fragments will then be cleaned up and sequenced using multiple primers in both directions to ensure adequate sequence coverage and accuracy. A dye terminator cycle sequencing reaction will be used to rebuild the amplified fragments incorporating dideoxynucleotides labelled with fluorescent dyes for sequence determination, and the library of fragments will be analysed using capillary sequencing. The data from this sequencing will be analysed using various software packages to align all the sequence reads and create a consensus for all sequenced regions. Mutations will be identified by comparing the sample consensus with known data
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of wild-type and mutant sequences. The advantage of this technique is that it can detect novel mutants; however the disadvantage of this technique is sensitivity in the context of a mixed sample- Sanger sequencing only works well when the tumour DNA makes up at least 25% of the total sample, though this can be improved by careful PCR design: product sizes of <150 bp to take account of DNA fragmentation in FFPE samples (Young et al., 2013).
1.4.4 Pyrosequencing
Pyrosequencing assays are designed to detect specific mutations, and in many labs are used alongside fragment length analysis to identify deletions and insertions. Pyrosequencing works by detecting the release of pyrophosphate molecules upon the incorporation of a specific nucleotide. A typical pyrosequencing protocol is similar to Sanger sequencing: DNA is extracted from the clinical sample and amplified by PCR. The PCR amplicons are then cleaned up and used as the template for the pyrosequencing reactions. This method is more sensitive than Sanger sequencing- the minimum level of tumour DNA in a wild-type sample is 5% (NICE, 2012). An example of one of the pyrosequencing assays available for EGFR mutant detection is the Qiagen Therascreen EGFR Pyro Kit (Qiagen, 2011).
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Figure 1.4.4 Diagram representing the chemistry of pyrosequencing technology. With each incorporation of a dNTP molecule, a pyrophosphate molecule is released, which in turn is catalysed by the sulphurylase enzyme to release ATP. The ATP is used as substrate by the luciferase enzyme to generate luciferin and light emission, which is detected by a camera. The strength of the light signal is proportional to amount of ATP present. Adapted from (Ahmadian et al., 2006).
1.4.5 Fragment Length Analysis
Fragment Length Analysis is a technique that distinguishes DNA fragments based on their length, therefore it is best employed for the detection of deletions and insertion events, as substitutions will have no effect on fragment size (Ellison et al., 2013). Extracted DNA samples are extracted, amplified by PCR and fluorescent dyes are incorporated. The
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amplicons are then combined with mixed size standards and analysed by electrophoresis. The fluorescence intensity is used to establish the fragment size and detect any insertions or deletions. In some cases, fragment length analysis has been shown to be more effective at detecting exon 19 deletions than direct sequencing (Pan et al., 2005).
1.4.6 Single Strand Conformation Polymorphism Analysis
Single Strand Conformation Polymorphism (SSCP) Analysis is a screening method that distinguishes sequence variations by changes in electrophoretic mobility differences. Extracted DNA is extracted and amplified by PCR. The amplicons are then denatured into single stranded DNA molecules. In single stranded form the DNA will spontaneously undergo three-dimensional folding that will create a unique structure depending on the nucleotide sequence. Single stranded molecules of differing nucleotide sequence will fold differently, and these differences in folding configuration can be assessed by gel electrophoresis (NICE, 2012).
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Figure 1.4.6. Diagram demonstrating the principle of Single Strand Confirmation Polymorphism (SSCP) Analysis. Adapted from (ThermoFisher, 2016). Mutant and wild-type single stranded amplicons will three dimensionally fold in a unique conformation which can be distinguished by gel electrophoresis.
1.4.7 High Resolution Melt Analysis
High Resolution Melt (Dubsky et al.) Analysis is a fast and cost effective screening method for all types of mutations in double stranded DNA e.g. polymorphisms and epigenetic changes. The method works by analysing the temperature at which different DNA molecules separate as temperature increases and can be combined with COLD-PCR (Co-amplification at Lower Denaturation temperature-PCR) and careful primer design to produce highly specific assays still in clinical diagnostic use (Pichler et al., 2009). Intercalating fluorescent dyes are incorporated into amplified DNA fragments in a PCR step before HRM analysis. The dyes fluoresce strongly when incorporated into double stranded DNA. During the HRM analysis, the increasing temperature causes strands to separate and fluorescence to drop suddenly. By analysing known wild-type samples alongside unknown or mutant samples, the difference in temperature at which fluorescence drops can identify mutant alleles. This
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technique can also distinguish heterozygous or homozygous mutants in the analysed amplicons (Pasay et al., 2008).
Figure 1.4.7. Diagram showing the melt curves from HRM analysis. The amplicon containing the mutant gives a different melt curve to the amplicon containing the wild-type, which is demonstrated by the change in fluorescence signal. Adapted from (Willmore-Payne et al., 2006).
1.4.8 Next Generation Sequencing
Next Generation Sequencing (NGS) is a relatively new technique, having recently revolutionised genetic analysis options available to molecular researchers. It has by far the broadest and deepest coverage of all the techniques described, but comes with some significant drawbacks. These drawbacks can be summarised as turnaround time and cost. Although NGS platforms vary, generally the library preparation can be very laborious, taking several days to complete before the actual sequence analysis can be run. Related to turnaround time is also the time and skill required for analysis, despite the fact that a lot of
Wild-type amplicon Mutant amplicon Fl u o resc ence
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the alignment and variant calling is done automatically, the final analysis can still be very involved, and some expertise is required for the initial setting up of the analysis parameters that the automated systems follow. Cost is also a major issue. The equipment, reagents and consumables for NGS are very expensive, and often only a limited number of patient samples can be analysed in any given run. There are multiplex options available to researchers using NGS systems; however the cost per patient still remains high (Cree et al., 2014) .