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  Table of Contents  
REVIEW ARTICLE
Year : 2017  |  Volume : 54  |  Issue : 5  |  Page : 9-14
 

Epidermal growth factor receptor mutation testing: From conventional to real-time diagnosis of lung cancer


1 Department of Medical Oncology, Apollo Speciality Hospital, Chennai, Tamil Nadu, India
2 MVR Cancer Centre and Research Institute, Kozhikode, Kerala, India

Date of Web Publication29-Dec-2017

Correspondence Address:
Dr. T Raja
Department of Medical Oncology, Apollo Speciality Hospital, Chennai, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijc.IJC_507_17

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 » Abstract 


Patients with non-small cell lung cancer (NSCLC) commonly harbor epidermal growth factor receptor (EGFR) mutation. Due to the complex disease pathology, early-stage diagnosis of patients with EGFR mutation is essential to make appropriate treatment decision. Tyrosine kinase inhibitors (TKIs) are commonly used for their treatment, but almost half of the patients with EGFR mutation do not respond to the available TKIs and develop acquired resistance owing to T790M mutation. The presence of T790M mutation also warrants a robust diagnostic method so as to allow clinicians to modify cancer treatment. Numerous diagnostic techniques for the detection of EGFR mutation, however, their performance and working profile variation necessitate a comparative evaluation for the selection of a better diagnostic method or an advanced combination of theirs. The present review compares various EGFR-mutation detection techniques such as Sanger sequencing, next-generation sequencing, and different polymerase chain reaction (PCR)-based methods. It also highlights the role of advanced PCR-based techniques, i.e., real-time or quantitative PCR and digital droplet PCR (ddPCR) for detecting EGFR mutations in NSCLC patients. ddPCR, when compared to other methods, shows enhanced sensitivity, superior reliability, and improved time and cost-effectiveness. Moreover, its ability to detect EGFR mutations including T790M, in both conventional (solid tissue biopsy samples) and nonconventional sample sources (blood, plasma, and urine samples), gives it an edge over other diagnostic techniques and support its integration in clinical practice setting.


Keywords: Digital droplet polymerase chain reaction, epidermal growth factor receptor, next-generation sequencing, quantitative polymerase chain reaction, Sanger sequencing, T790M mutation


How to cite this article:
Raja T, Warrier N K. Epidermal growth factor receptor mutation testing: From conventional to real-time diagnosis of lung cancer. Indian J Cancer 2017;54, Suppl S1:9-14

How to cite this URL:
Raja T, Warrier N K. Epidermal growth factor receptor mutation testing: From conventional to real-time diagnosis of lung cancer. Indian J Cancer [serial online] 2017 [cited 2018 Jan 21];54, Suppl S1:9-14. Available from: http://www.indianjcancer.com/text.asp?2017/54/5/9/221921





 » Introduction Top


Non-small cell lung cancer (NSCLC), comprising of 80% of lung cancer cases, is considered to be heterogeneous in nature and is difficult to treat due to its complex pathology.[1],[2],[3],[4],[5] The presence of epidermal growth factor receptor (EGFR) mutation is commonly observed in patients with NSCLC.[4] However, there are not many studies available which have reported the prevalence of EGFR mutations in patients with NSCLC in India. In 2011, Sahoo et al. reported EGFR mutation to be present in 52% of NSCLC patients (higher in females as compared to males).[6] Similarly, a study by Veldore et al. (2013) reported that almost 41% of NSCLC patients have EGFR mutation and the four major oncogenic driver mutations are exon 18 (2%), 19 (24%), 20 (2%). and 21 (13%).[4] Noronha et al. also studied the prevalence of EGFR mutations in NSCLC patients in India and reported that about 35% of studied population had EGFR mutations, with a majority being females (48%) and nonsmokers (81%).[7] In contrast, another Indian study reported that 23% of NSCLC patients harbor an EGFR mutation.[8]

Conventional therapies such as systemic chemotherapy have been the basis of treating advanced NSCLC. However, they are associated with uncertain benefits and numerous adverse effects.[9] This led to the development of tyrosine kinase inhibitors (TKIs) which specifically target EGFR mutation in NSCLCs patients. However, majority of patients with EGFR mutation are found to be resistant (primary resistance) or gradually develop resistance (acquired resistance) after EGFR-TKI therapy. In NSCLC patients with acquired resistance, T790M mutation is found in approximately 50%–60% of the cases.[10] Therefore, along with sensitizing EGFR mutation in exons 19 and 21, identifying the resistant mutation T790M is also important and critical for treatment-related decision-making.[11]

Performing EGFR molecular testing in patients with lung cancer enables the physicians in determining and modifying the treatment regimen.[4],[12] Moreover, various randomized trials also support the significance of performing EGFR mutation testing before the initiation of NSCLC treatment.[13] Several traditional methods such as immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and denaturing high-performance liquid chromatography (DHPLC) are available for EGFR mutation, but they provide limited information. More sensitive methods such as polymerase chain reaction (PCR)-based methods (reverse transcriptase-PCR [RT-PCR], real-time PCR, quantitative PCR [qPCR], allele-specific PCR, etc.) are also available. In India, PCR-based methods and Sanger sequencing (SS) is used in about 20%–50% of NSCLC cancer cases with EGFR mutation.[14] Noronha et al. (n = 111) and Choughule et al. (n = 907) used TaqMan-based qPCR technique and sequencing method, whereas Veldore et al. (n = 1036) and Sahoo et al. (n = 220) used Scorpion probe-based amplified refractory mutation system (ARMS)-PCR technique for EGFR mutation testing in several subsets of lung cancer patients.[4],[6],[8]

However, there lies ambiguity about the working profile and performances (sensitivity, cost-effectiveness, sample type, quantity required for testing, advantages, and disadvantages) of the EGFR mutation testing techniques, which warrants a comparative evaluation of the different methods so that a better diagnostic method or a combination of methods can be selected.[15],[16] Moreover, all these factors would also influence clinician's decision in selecting a technique which is sensitive, cost–effective, and can detect all varied EGFR mutations from limited amount of samples in advanced stage NSCLC patients so as to shorten the diagnostic time and expedite treatment decisions.[14],[17],[18] The present review highlights various techniques used to detect EGFR mutation along with the advantages, disadvantages, and their comparative data. The review majorly focuses on the role of advanced PCR-based techniques, i.e., qPCR and digital droplet PCR (ddPCR) in the detection of EGFR mutations in NSCLC patients.


 » Samples Used for Epidermal Growth Factor Receptor Mutation Testing Top


The source of tumor material significantly plays an important role in selecting the mutation testing technique.[17] Most of the diagnostic tests use formalin-fixed, paraffin-embedded tissue (FFPET) specimens. In cases where tissue samples are unavailable or obtained in suboptimal quantity, cytology samples are used. This involves less invasive method of sample collection.[19],[20],[21] Recently, another repeatable, less invasive sampling source has been introduced, i.e., serum, which can be used to assess EGFR mutations in patients where surgery is not feasible.[22] Methods such as IHC, FISH, DHPLC, SS, and qPCR use solid tissue biopsy samples, whereas the techniques using samples from nonconventional sources, such as blood, plasma, and urine samples, apart from tissue biopsy samples, are ddPCR and allele-specific PCR.[23],[24]


 » Techniques Used for Epidermal Growth Factor Receptor Mutation Testing Top


The diagnosis of EGFR mutation involves the use of many screening methods. According to molecular scientists, detection of EGFR mutation with just a single method misjudges EGFR-related abnormalities; therefore, the screening methods should be used in combinations.[25] [Figure 1] presents the performance of various EGFR mutation testing techniques. The EGFR mutation testing techniques can be broadly classified as follows:
Figure 1: Sensitivity of EGFR mutation techniques

Click here to view


  1. SS
  2. Next-generation sequencing (NGS)
  3. PCR-based methods.


Sanger sequencing

SS has been considered as the gold standard to detect EGFR mutations since the 1970s and still maintains its popularity in EGFR mutation testing and is one of the commonly used clinical methods for detection of known and novel EGFR mutations in diagnostic laboratories.[15],[26] It has also been observed that, SS, if performed efficiently, can still be considered as the gold standard.[17] To attain accurate results, SS can be used in combination with DHPLC and different PCR-based methods.[26]

However, the disadvantages/drawbacks associated with SS have also limited its popularity in the clinical setting. It is a multiple step process and involves the extraction of DNA followed by DNA's PCR-based amplification and sequencing and data interpretation. All these make it a time-consuming process. Subsequently, its suboptimal sensitivity and requirement of large sample amount for analysis add to the reasons for its limited use. Additionally, the tumor sample needs to have more than 25% of mutant DNA for optimal mutation detection results.[15]

Next-generation sequencing

NGS, also known as massive parallel sequencing, has gradually replaced the traditional methods of EGFR mutation testing (SS) due to much higher sensitivity and the ability to detect mutation in samples with low allele frequency.[27],[28] It is an accessible, affordable, and an acceptable method of detecting oncogenic mutations.[29] As the name suggests, the technique analyzes numerous DNA molecules simultaneously and that too at a lower cost.[30] It has gained popularity due to its ability to detect all the three types of clinically relevant genetic alterations, i.e., alteration in single nucleotide, copy number changes, and genetic rearrangements in multiple genes in a single run.[31]

NGS, using Ion Torrent technology for detection of gene mutation in lung adenocarcinoma using fine-needle aspiration cytology samples, identified multiple gene mutations at one time with a sample DNA amount as low as 10 ng DNA.[32] Another study comparing the same platform (NGS with Ion Torrent platform using the Ion AmpliSeq Cancer Hotspot Panel v2) with qPCR (TaqMan) and SNaPshot assay combined with fragment analysis reported the identification of 15/64 EGFR mutations by qPCR and 11/64 EGFR mutations by SNaPshot assay, whereas NGS identified 10 mutations that were identified by the SNaPshot/qPCR panel. In addition, other three insertions, five point mutations, and 58 silent variants were also detected using NGS technique.[33] Another study reported the detection of 3 cases out of 14 biopsy samples by SS and 6 by NGS (454 GS Junior Next-Generation sequencer, Roche Diagnostics, Mannheim, Germany); 11 cases out of 66 cytology samples by SS and 18 by NGS. It also emphasized several advantages of NGS as compared to SS such as high analytical sensitivity, comprehensive screening of the target region, high throughput, and semi-quantitative evaluation of the mutated allele. Moreover, SS was not able to detect any mutation in samples containing ≤40% of mutated material, whereas NGS was able to detect samples with 5% mutation also.[34]

Despite the advantages of NGS to easily identify samples with high tumor content along with creating an elaborative mutational profile and identifying novel chromosomal rearrangements and copy number alterations, the samples with low tumor content warrant careful data analysis which is laborious and increases the overall cost.[33]

Polymerase chain reaction-based methods

PCR is used to detect EGFR gene mutation by exponential amplification of a particular DNA sequence. As compared to SS, it is a rapid method, as well as the initial step to identify common gene mutations (L858R and the most common exon 19 deletion). It can even detect mutation in low frequency (1%) mutant alleles. However, its poor selectivity in amplifying low-level mutations in a wild-type background and its ability to detect only the prespecified mutations for which it is designed are the major loopholes associated with PCR. Moreover, PCR cannot detect DNA extracted from FFPETs.[35],[36] Therefore, a combination of SS and PCR has been considered as the second most frequently used method for EGFR mutation testing.[13] To achieve better outcomes by identifying rare genetic mutations as well as for clinical decision-making, PCR technique has undergone modifications such as mutant-enriched PCR and RT-PCR.[37],[38],[39]

Co-amplification at lower denaturation temperature-polymerase chain reaction

Co-amplification at lower denaturation temperature-PCR (COLD-PCR) has the tendency to enhance minority alleles exclusively in a mixture of wild-type and mutation-containing sequence regardless of the type of mutation or sequence position, thereby improving the sensitivity of the technique. Li et al. replaced conventional PCR with COLD-PCR to evaluate the effect of mutation enrichment on the performance of Sanger dideoxy-terminator sequencing in a patient with NSCLC and reported a 100-fold improvement in sensitivity while detecting mutation. The method was also able to identify new/missed mutations in the genes encoding p53, KRAS, and EGFR.[39]

Mutant-enriched polymerase chain reaction

It is a rapid, sensitive as well as a reliable method which has the capability to detect one single mutated EGFR gene (preferably in exons 19 and 21) out of 100s of genes found in samples obtained from biopsies, pleural fluid, and surgically resected tissues.[40],[41] The major disadvantage associated with mutant-enriched PCR is its inability to detect minor alterations and obtaining false-positive results.[18]

Amplified refractory mutation system

ARMS is a simple PCR-based detection method used to detect mutation in both tumor tissues as and plasma/serum samples. It is considered better than SS due to high sensitivity and high-throughput analysis of clinical samples. However, it is unable to detect all activating EGFR mutations.[42],[43] It is more sensitive than DHPLC with highest capacity to detect mutations; however, DHPLC is comparatively less expensive and technically easier to perform.[26],[42],[43] ARMS is better validated, ready for use, and quality controlled as compared to other laboratory methods.[20] As per Ellison et al., ARMS can detect mutation in samples with 1% mutant gene, thereby showing a higher sensitivity (0.1%–1%).[44] The major disadvantage associated with ARMS is presenting false-positive results. This can be avoided using better techniques such as microfluidics digital PCR.[22]

Quantitative polymerase chain reaction

qPCR, also known as quantitative PCR, is a method used to measure the quantity of a PCR product in real time and is therefore also called real-time PCR. It is used to determine the number of copies of a DNA sequence in a sample. qPCR-based platforms such as the cobas ® EGFR mutation test (Roche Molecular Systems, Pleasanton, CA, USA) and therascreen EGFR RGQ PCR Kit (Qiagen, Hilden, Germany) are specially designed to identify EGFR mutations. cobas ® test can detect 41 EGFR mutations in a maximum of 30 samples per run with matched or superior performance as compared to SS, whereas therascreen Kit has the ability to detect 29 EGFR mutations in upto 7 samples per run and is attributed to 100% sensitivity and specificity as compared to SS.[45],[46],[47]

qPCR uses fluorescent dyes, such as SYBR Green, EvaGreen, or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time.[48] qPCR using TaqMan probe allows amplification of PCR as well as detection of probe in one tube only which avoids the use of ethidium bromide staining, thereby avoiding cross-contamination coupled with other advantages such as saving time and as an easy diagnostic method for clinical practice.[49] A study by Zhou et al. evaluated qPCR using TaqMan probes which identified 8 somatic point mutations and 13 deletions in the tyrosine kinase domain of the EGFR gene in 80 NSCLC patients with 100% sensitivity and specificity.[50] Another method used by Sasaki et al., i.e., qPCR with mutation-specific sensor and anchor probes (double-stranded PCR product formation assay performed by Light Cycler) measured fluorescence of the SYBR Green dye (instead of ethidium bromide) for detection of double-stranded PCR product formation.[49] The LightCycler qPCR used to detect EGFR genotyping in exons 18, 19, and 21 of NSCLC tumor tissues obtained from 118 patients identified 9 missense mutations in exon 21, 11 deletions in exon 19, and 2 missense mutations in exon 18.[49] With qPCR, the wild-type tumors can be characterized in a very small amount of sample. However, distinction between the different types of deletions in exon 19 is not possible.[17] As compared to conventional PCR-based methods, qPCR is a reliable method with high sensitivity (100% for exons 19 and 21).[17] However, this method is unable to detect unknown mutations.[11]

Digital droplet polymerase chain reaction

ddPCR is unparalleled in terms of sensitivity and precision due to its ability to quantify EGFR mutations at single-molecule level. It is sensitive enough to detect low rate of mutation in tumor sample.[22],[51] In addition to tumor cells, ddPCR can detect EGFR mutation in DNA obtained from blood plasma and urine samples.[23] In ddPCR, a water-in-oil emulsion is prepared to produce 20,000 nanoliter-sized droplets. Each droplet containing some/no template copies undergoes further amplifications. To analyze each droplet, a fluorescence detector is used, while the Poisson modeling equation is applied to measure the copy numbers of the target sequences.[52]

Among all the PCR techniques used, qPCR and ddPCR are found to be the most reliable and most commonly used techniques in pathology departments. However, ddPCR is found to be better than qPCR due to several reasons. It is an easy, simple, and cost-effective method which is highly sensitive (can detect 1 mutated DNA/20,000 copies of normal DNA). The major advantage of this technique is in the detection of mutation from FFPE samples as well as tumor cell-free DNA specimens.[24] Another major difference between the two lies in their nature of detection. qPCR requires a standard curve and is semi-quantitative in nature which makes it error prone. Moreover, it just informs about the negative/positive status of EGFR gene mutation. On the other side, ddPCR is more sensitive, does not require a standard curve, and has the capability to detect rare mutation sequences as well.[11] It has been reported that ARMS-qPCR has the capability to precisely analyze samples at low mutation rates (1%), whereas ddPCR can detect samples at much lower rates (0.1%–5%). A study, by Zhang et al., compared the performance of ARMS-qPCR and ddPCR in the detection of EGFR mutation and reported ddPCR to be a better technique for detecting samples with low mutation rates. ARMS-qPCR method detected the 6000 copies of plasmid samples with 1%–5% mutation rates, whereas ddPCR detected them with 0.1%–5% mutation rates (0.1%, average 6 copies; 0.5%, 24 copies; 1%, 57 copies; and 5%, 398 copies).[52]

Studies suggest ddPCR to be a reliable method for the detection of T790M mutation. A study conducted by Thress et al. compared the ability of different technology platforms, i.e., two nondigital platforms (cobas ® EGFR Mutation Test and therascreen™ EGFR ARMS-PCR) and two digital platforms (ddPCR and BEAMing digital PCR [dPCR]) to detect EGFR mutations, including T790M using circulating tumor DNA (ctDNA) samples obtained from advanced NSCLC patients. A higher sensitivity was observed using digital platforms (ddPCR: 71%; BEAMing dPCR: 71%) for detection of T790M mutation as compared to the nondigital platforms (cobas ® EGFR Mutation Test: 41% and therascreen™ EGFR ARMS-PCR: 29%).[53] Another study comparing the performance of ARMS-qPCR and ddPCR for detection of T790M mutation reported that the sample containing T790M mutation was identified as EGFR wild type by ARMS-qPCR, while ddPCR clearly revealed it as EGFR T790M mutation with seven copies of mutant alleles in a background of 6000 wild-type copies.[52]

Others

A sensitive and rapid PCR method used to detect 11 types of EGFR mutations is peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp.[54] The major advantage associated with this method is that the PNA and LNA clamp primers bear wild-type and mutant sequences, respectively. The mutant LNA probes are inhibited by PNA clamp primers which increase the specificity of detection. The sensitivity is also high as this method can detect EGFR mutations in 1000s of samples.[55] PCR-strand conformation polymorphism (PCR-SSCP) is another sensitive method used by Marchetti et al.[56] It was found to be better than SS in terms of sensitivity without showing false-positive or false-negative results.[57]

In a study by Simonetti et al., deletions in exon 19 were determined by length analysis after PCR amplification, whereas 21 point mutations were detected using TaqMan PCR assay.[58]

Nakamura et al. developed two highly sensitive and reliable methods for detection of EGFR mutation in wild inhibiting PCR and quenching probe system (WIP-QP) and mutation-biased PCR and quenching probe system (MBP-QP) for exon 21 L858R mutation and exon 19 deletion, respectively.[59]

RT-PCR involves transformation of RNA into cDNA for amplification. It is a rapid method widely used to determine the expression of a gene and does not require post-PCR sample handling which prevents potential PCR product contamination. This results in it being a much faster and higher throughput assay.[60]

Other methods

Other methods used for EGFR mutation screening involve fragment length analysis [20] and massively parallel sequencing.[61] A rapid, sensitive, and extensive molecular characterization of tumors (using methods such as Sequenom MassARRAY ®, SNaPshot, and arrays of mutation-specific PCR assays, e.g., q-Biomarker Somatic Mutation PCR Array) is under research so as to select the most appropriate therapy.[20]


 » Conclusion Top


There are different diagnostic methods used to detect EGFR mutation in patients with NSCLC including SS, NGS, and PCR-based techniques. Due to high variability in their performances, all of them cannot be used in clinical settings. Out of all the commonly used methods, qPCR and ddPCR are more reliable methods for detecting common EGFR mutations as well TKI-resistant mutation, i.e., T790M in patients with NSCLC due to their high sensitivity and specificity. Moreover, the benefit associated with ddPCR and the use of tissue, urine as well as blood sample for detection make it more compatible for use. Hence, these methods could be considered by diagnostic centers to detect EGFR mutation so as to assist the physicians for appropriate treatment-related decision-making.

Acknowledgments

The authors acknowledge AstraZeneca Pharma India Ltd and Jeevan Scientific Technology Ltd for medical writing and editing support.

Financial support and sponsorship

Financial support to authors - Nil.

The supplement issue in which this article has been published has been sponsored by AstraZeneca Pharma India Ltd.

Conflicts of interest

There are no conflicts of interest.



 
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