Recent Validations of EGFR testing Methods

The three previous sections (1.8.1- 3) give an overview of the literature covering various aspects of EGFR mutation detection up to late 2014 when this project was in its early stages. Since 2014, there have been a number of publications describing validation studies of new diagnostic methods based on many of the techniques previously described. From 2015 to present there are six publications in particular that present interesting studies, some of

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which were diagnostic validations. Of the six studies: three used PCR based techniques (Vliegen et al., 2015; Malapelle et al., 2016; Zhang et al., 2015b) , one used pyrosequencing (Xie et al., 2015), one used NGS (Fujita et al., 2015), and one used MALDI-TOF (Matrix- Assisted Laser Desorption/Ionization Time Of Flight) (Su et al., 2016).

Vliegen et al. (Vliegen et al., 2015) validated a combined locked nucleic acid (LNA) PCR/ Sanger sequencing assay for the detection of EGFR exon 18 mutations (G719A/S/C) and exon 19 deletions in DNA extracted from FFPE tissues samples. This assay consisted of two steps. First the extracted DNA samples were amplified using the LNA PCR assay. The LNA molecules blocked amplification of the wild-type sequences, therefore enriching the amplicons for the mutant alleles (if present). The amplified products were then sequenced using conventional Sanger sequencing. The results from the LNA/ Sanger assay were compared retrospectively to the original diagnostic results using Fragment Length Analysis and The Qiagen Therascreen EGFR assay. The LNA/ Sanger assay demonstrated a high degree of concordance with the Therascreen results, 100% of samples tested on both techniques gave the same results. The assay also displayed a high level of sensitivity with a LoD of 3.13% tumour cell content. The study concluded that the LNA/ Sanger assay achieved equivalent accuracy, whilst being 25- 50% cheaper than the Therascreen assay. The disadvantage however was that the LNA/ Sanger assay required more hands on time, and in its present form does not cover all the most common EGFR mutations.

The study by Malapelle et al. (Malapelle et al., 2016) aimed to address the problem of the difficulties encountered when attempting to detect EGFR mutations from cytological smear samples which are small samples and thus contain a very low number of tumour cells (and therefore a low concentration of tumour DNA). Their approached involved the use of a digital PCR (dPCR) technique, designed to detect EGFR exon 19 deletions and the L858R mutation.

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The dPCR assay only required 2 ng of input DNA, and can detect mutation rates at 0.1% (Zhang et al., 2015a) so therefore should be well suited to low DNA concentration samples. The results from this study showed the dPCR technique to be sensitive and accurate. The dPCR was tested down to 1% in LoD assays and a majority (19/20) of cytology samples tested gave the same results as previous testing. In one case the dPCR detected an additional L858R mutation which was not detected by the previously used assays (Fragment Length Analysis and TaqMan PCR). The additional mutant was confirmed by cloning and sequencing. This study demonstrated that dPCR is a suitable technique for the detection of EGFR mutants, and offers a considerable advantage in terms of low DNA requirement and robustness with low DNA concentration/ quality samples. In a previous study, this group reported that 16.2% of the cytological samples received for analysis had to be rejected due to insufficient cellularity (Malapelle et al., 2013). The improved sensitivity of dPCR may reduce this rejection rate, as well as reduce the service turnaround time and assay failure rate.

Zhang et al (Zhang et al., 2015b) performed a study using the SNaPshot® technique to detect oncogenic mutations in a number of genes including EGFR, KRAS and BRAF, as well as KIT and PIK3CA. This article is slightly different to others reviewed here, as the clinical context of this article is nasopharyngeal carcinoma (NPC), however this does not impact on the technical aspects of mutation detection assays. Also EGFR is considered to be an important mutation in NPC, with up to an 80% prevalence of EGFR overexpression in NPC primary biopsies (Pan et al., 2008). EGFR mutations however are rare in NPC, at 0-1% prevalence (Lee et al., 2006). 70 fresh frozen tissue samples from NPC patients were extracted and analysed using the SNaPshot® assay. The result of mutation detection from these samples was compared to clinicopathologic data. Of the 70 samples, 12 (17.1%) tested positive for the presence for hotspot mutations in at least one of the five oncogenes analysed (3 in EGFR). These results were consistent with the findings of a previous study (Jiang et al., 2014). The SNaPshot® assay

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passed all assay controls, however there was no comparator method used to compare the mutation results against. No correlation was found between the mutation status and the clinicopathologic data for each patient.

Xie et al. (Xie et al., 2015) performed a large study, using pyrosequencing to analyse FFPE tissue samples from NSCLC and CRC patients. In the total the study analysed 494 and 1099 tissue samples for NSCLC and CRC respectively. Extracts from cell lines were also used to perform LoD experiments. The pyrosequencing technique demonstrated the ability to detect 2% mutant alleles in the wild-type background. Sanger sequencing was used to confirm the results from the LoD experiments with cell lines. In the NSCLC samples, the pyrosequencing detected EGFR mutations in 176 of 494 (35.6%). In the 1099 CRC samples, 437 samples contained mutations (39.8%). Sanger sequencing was used to confirm the results generated by the pyrosequencing. These results show a very high prevalence of EGFR and KRAS mutations amongst NSCLC and CRC patients, and that pyrosequencing appears to be a sensitive and accurate means of detecting mutations in FFPE tissue. However the study reported that in samples tested using both Sanger sequencing and pyrosequencing, pyrosequencing detected more mutations, which is most likely due to the relatively low sensitivity of direct sequencing. As a result the pyrosequencing technique used in routine clinical service at the author’s resident hospital.

The study by Fujita et al. (Fujita et al., 2015) was a validation study of Next Generation Sequencing assay for the detection of oncogenic mutations from FFPE tissue biopsy samples from NSCLC patients. The NGS assay ran on the Ion Torrent platform, utilising the Ion AmpliSeq™ Cancer Hotspot Panel version 2, which is designed to analyse 50 cancer hotspot genes, including EGFR and KRAS. 21 tissue samples were analysed using the Ion Torrent assay. A cycleave PCR assay (Yatabe et al., 2006) and Sanger sequencing were used to verify

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the results. Comparison of the results showed the mutations detected by the NGS platform matched the results from the other assays in all cases. In addition, in one case a low frequency T790M mutation was identified in 53 of 900 reads (5.9%) for that amplicon, possibly demonstrating the emergence of a resistant subpopulation in that patients tumour. This subpopulation would not be detectable by Sanger sequencing and may not have been detectable by the cycleave PCR assay. This study showed the NGS assay for the accurate, sensitive and practical for use in the clinical setting, especially when there is limited material for molecular analysis.

The final study in this section is by Su et al. (Su et al., 2016) and is slightly unusual compared to the other studies as the main mutation detection technique used was matrix-assisted- laser-desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) rather than PCR or sequencing. This article is an interesting contrast to other approaches, but still requires the extraction and amplification of DNA and thus has a molecular biology component similar to other studies. Briefly, the procedure involved extraction of the sample DNA, amplification of the target region, treatment with SAP (shrimp alkaline phosphatase), single nucleotide extension with mutation specific probe, then MALDI-TOF MS analysis. The technique was tested and optimised using mutant and wild-type cell lines, achieving a LoD of 1%. Analytical sensitivity and specificity were tested with a panel of clinical samples. Sanger sequencing was used as the comparator method. The test achieved 100% analytical sensitivity and specificity, so the technique went into routine use. In the course of the period covered by the study, 8147 lung adenocarcinoma samples were tested with this technique, of the failure rate was only 0.1% (n= 5). This study shows an interesting alternative approach to EGFR mutation detection. The only technical issue presented was that the technique occasionally detect positive mutants in their wild-type PBMC (peripheral blood mononuclear

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cell) DNA which was attributed to background signal, which highlights the need to establish accurate thresholds for clinical diagnostic use.