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  In this article
 »  Abstract
 » Introduction
 »  Oncogenic Driver...
 »  Epidermal Growth...
 »  Acquired Resista...
 »  Epidermal Growth...
 »  Molecular Tests ...
 »  Epidermal Growth...
 » Screening Methods
 » Targeted Methods
 »  Challenges With ...
 »  Tumor Heterogene...
 » Liquid Biopsy
 »  Circulating Cell...
 »  Circulating Tumo...
 » Exosomes
 »  Tumor-Educated P...
 »  Clinical Applica...
 »  Requirement of S...
 »  Plasma Genotypin...
 »  Droplet Digital ...
 »  Amplification Re...
 »  Beads, Emulsion,...
 »  Next-Generation ...
 »  Plasma Genotypin...
 »  Plasma Genotypin...
 »  Cross-Platform C...
 »  Challenges With ...
 » Conclusion
 »  References
 »  Article Figures
 »  Article Tables

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  Table of Contents  
Year : 2017  |  Volume : 54  |  Issue : 5  |  Page : 45-54

Epidermal growth factor receptor T790M testing in progressed lung cancer: A review of sensitive methods for analysis of tissue and liquid biopsy samples

1 Tata Memorial Centre, Medical Oncology-Molecular Laboratory, Mumbai, Maharashtra, India
2 National Diagnostics Head, Medical Affairs, Astra Zeneca Pharma Limited, Bengaluru, Karnataka, India

Date of Web Publication29-Dec-2017

Correspondence Address:
Dr. A Chougule
Tata Memorial Centre, Medical Oncology-Molecular Laboratory, Mumbai, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijc.IJC_540_17

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 » Abstract 

Lung cancer is one of the major causes of mortality worldwide and is on the rise in India. The identification of epidermal growth factor receptor (EGFR) mutations in nonsmall cell lung cancer (NSCLC) has paved the way for personalized therapy in lung cancer with EGFR-tyrosine kinase inhibitors (TKIs). Despite the proven efficacy of EGFR-TKIs in patients harboring EGFR mutations, their clinical utility is limited by the development of acquired resistance mechanisms by the tumor cells. T790M mutation accounts for 60% of all resistance mechanisms to EGFR TKIs and is responsible for treatment failure with first- and second-generation TKIs. With the development of novel therapeutic agents such as osimertinib to overcome this resistance mechanism, it is essential to detect patients harboring T790M mutation. There are several limitations with the use of tissue biopsy specimens for molecular testing such as poor quality and quantity of sample, tumor heterogeneity, occurrence of complications, and issues with repeat biopsy. Liquid biopsy offers a noninvasive approach that can be used for diagnostic purposes as well as for monitoring treatment response and evaluation of resistance mechanisms. This review focuses on the methods for molecular testing of tissue and liquid biopsy specimens for EGFR mutations, particularly EGFR T790M mutation.

Keywords: EGFR-tyrosine kinase inhibitors, liquid biopsy, nonsmall cell lung cancer, T790M mutation

How to cite this article:
Chougule A, Basak S. Epidermal growth factor receptor T790M testing in progressed lung cancer: A review of sensitive methods for analysis of tissue and liquid biopsy samples. Indian J Cancer 2017;54, Suppl S1:45-54

How to cite this URL:
Chougule A, Basak S. Epidermal growth factor receptor T790M testing in progressed lung cancer: A review of sensitive methods for analysis of tissue and liquid biopsy samples. Indian J Cancer [serial online] 2017 [cited 2022 Jul 4];54, Suppl S1:45-54. Available from:

 » Introduction Top

The incidence of lung cancer is on the rise in India. Among males, the estimated number of new cases of lung cancer was 62,650 in 2011 which is projected to rise to 103,360 new cases by 2026. Among females, the number of new cases is expected to reach 35,436 in 2026 from the estimated number of 21,590 in 2011.[1] The GLOBOCAN 2012 reported an estimated number of 70,275 lung cancer cases in all ages and both sexes in India.[2] Lung cancer accounts for 9.3% of all cancer-related mortality in both sexes and is the leading cause of deaths due to cancer in men.[3] The single most important risk factor for lung cancer is tobacco smoking.[2]

A changing trend has been observed in the incidence of histological type of nonsmall cell lung cancer (NSCLC), with an increase in the incidence of adenocarcinoma over the earlier predominant squamous cell type of cancer.[3],[4] With the availability of targeted drug therapy and proper selection of patients by molecular testing, there has been an increase in the overall survival in patients with lung cancer.[5] The molecular profiling of lung cancer has led to the identification of a number of oncogenic drivers which can help to tailor the treatment in patients harboring these mutations.[3] The frequency of epidermal growth factor receptor (EGFR) mutation varies from 20% to 40% in Indian lung cancer patients.[6],[7],[8] This review focuses on the methods for analysis of tissue and liquid biopsy specimens for EGFR mutations, particularly EGFR T790M mutation.

 » Oncogenic Drivers in Non-Small Cell Lung Cancer Top

The discovery of EGFR mutations in NSCLC has paved the way for genotype-directed, biomarker-driven therapy in lung cancer. Molecular profiling has led to the identification of a number of genetic alterations which highlight the interpatient as well as intratumor heterogeneity in lung cancer. The targetable somatic mutations that drive disease progression in lung adenocarcinoma include EGFR (10%–15% in frequency), BRAF, PIK3CA, and HER2 mutations (all three mutations are <5% in frequency). Anaplastic lymphoma kinase (ALK), ROS1, and RET rearrangements are the actionable genetic rearrangements in this type of NSCLC.[9]

Routine molecular profiling for EGFR mutations, ALK rearrangements, as well as HER2 (ERBB2), KRAS, BRAF, and PIK3CA mutations in patients with advanced NSCLC is feasible and offers clinical benefit. The overall survival is prolonged by 4.7 months in the presence of a genetic alteration than in the absence of genetic alteration.[10] Several studies conducted in India also highlight the role of mutational profiling in patients with advanced NSCLC.[11],[12]

 » Epidermal Growth Factor Receptor Mutation Top

Genotyping of EGFR tyrosine kinase domain has led to the identification of EGFR-sensitizing mutations as shown in [Figure 1].[13] The most common mutation is an in-frame deletion around the LREA motif of exon 19 (E746-A750) which comprises 50% of all EGFR-sensitizing mutation. The L858R substitution in exon 21 and missense mutations in exon 18 that affect the G719 residue constitute around 45% and 5% of all EGFR-sensitizing mutations, respectively.[9],[13] Other less common mutations include in-frame insertions within exon 20, exon 21 L861Q mutation, and in-frame exon 19 insertions.[14] EGFR mutations occur more frequently in females with NSCLC, in East Asian lung cancer patients, lung adenocarcinoma and in non or light smokers.[15]
Figure 1: EGFR mutations in EGFR kinase domain. EGFR = Epidermal growth factor receptor

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These sensitizing mutations activate EGFR signaling pathway in the absence of a ligand.[13] They change the equilibrium of kinase from inactive to active conformation, thus activating pro-survival and antiapoptotic downstream signaling pathways. These mutant cells are thus dependent on EGFR receptor for their survival which forms the basis for the use of EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutant tumors.[14] Exon 20 mutation results in a poorer overall survival prognosis compared to EGFR- and ALK-negative patients and patients harboring EGFR TKI-sensitizing activating mutations. The incidence of de novo exon 20 insertions is 3.4%, and different types of exon mutations have reported different outcomes.[16]

 » Acquired Resistance to Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors Top

A schematic representation of mechanisms of acquired resistance to EGFR-TKIs leading to treatment failure is shown in [Figure 2].[17] These include central nervous system (CNS) sanctuary as a result of poor penetration of drug into CNS in isolated CNS disease progression, target alteration (EGFR T790M mutation), and activation of bypass pathways (MET amplification, HER2 amplification) that overcome the inhibition of signaling pathway by EGFR-TKIs, increased expression of HER3, insulin growth factor-1 receptor (IGF-1R), and AXL tyrosine kinase, which lead to downstream pathway activation independent of EGFR activation and histological transformation of tumor, making it less sensitive to TKI therapy.[14],[17],[18]
Figure 2: Mechanisms of acquired resistance to EGFR TKIs. CNS = Central nervous system; ampl = Amplification; EGFR = Epidermal growth factor receptor; EMT = Epithelial to mesenchymal transition; Star indicates mutation

Click here to view

 » Epidermal Growth Factor Receptor T790m Mutation Top

The EGFR T790M mutation (substitution of threonine 790 with methionine) in exon 20 as shown in [Figure 1] is a clinically actionable resistance mechanism which accounts for 60% of all resistance mechanisms to EGFR-TKIs.[17],[18],[19] This gatekeeper mutation restores ATP affinity in EGFR-mutant cells, thus reducing the potency of ATP-competitive TKIs in EGFR-mutant NSCLC. It closes the therapeutic window that was available for first-generation EGFR-TKIs.[19] In addition, EGFR-mutant tumors with T790M mutation do not respond to second-generation TKI such as afatinib.[14] T790M mutation is extremely rare in tumors not treated with first-generation TKIs. It can occur de novo during treatment with TKIs, or subclones with this mutation may preexist at a low frequency in the tumor. Upon treatment with gefitinib or erlotinib, these subclones become apparent as they are resistant to treatment. T790M mutation can also affect substrate specificity as well as kinase activity of EGFR-mutant cells, leading to cell proliferation and survival.[20] Patients harboring T790M mutation have a relatively favorable prognosis; T790M mutation is an important prognostic and mechanistic biomarker which should be routinely assessed in patients with acquired resistance.[21]

 » Molecular Tests in Nonsmall Cell Lung Cancer Top

Single-gene molecular diagnostic assays for driver alterations in lung cancer include nucleotide sequencing and polymerase chain reaction (PCR)-based methods. Multiplex hotspot mutational testing such as a SNaPshot assay and Sequenom assay and multiplex sizing assays are examples of multiplex tests that enable comprehensive genotyping of lung cancer. Next-generation sequencing (NGS) or massively parallel high-throughput screening has the ability to sequence changes in the entire length of the gene of interest, covering both hotspot and nonhotspot regions.[22]

 » Epidermal Growth Factor Receptor Mutation Testing Using Tissue/cytological Samples in Lung Cancer Top

The molecular methods for EGFR mutation testing can be broadly categorized as screening and targeted methods. In contrast to the targeted methods, screening methods are more time-consuming, less sensitive, often need tumor cell enrichment, and require experienced personnel for their execution. However, rare mutations may be missed out in targeted assays, and reagents used for testing are expensive compared with screening methods.[23]

 » Screening Methods Top

Sanger or direct sequencing can detect a wide range of genetic alterations such as insertion, duplication, single nucleotide variants, and deletions. It requires only 5–10 ng of DNA but has low sensitivity. The mutant variant will be detected only if it constitutes at least 20% of the total tumor DNA. High-resolution melt (HRM) analysis is a sensitive technique that is used to identify samples with mutation. Subsequently, sequencing will be required to detect specific mutation. It is an inexpensive and rapid screening method.[24] HRM analysis, as a screening method, showed 100% sensitivity and 90% specificity in the detection of EGFR mutation from formalin-fixed paraffin-embedded (FFPE) NSCLC samples.[25]

Pyrosequencing or sequencing by synthesis can detect mutations in as low as 5% of tumor samples and is more sensitive than Sanger sequencing. It is, however, less effective in detecting structural chromosomal alterations and changes in gene copy number. NGS or massively parallel sequencing utilizes high-throughput screening to identify multiple genes of interest from limited tissue sample (as low as 5% of tumor material). Unlike targeted assays, it can be used to detect any mutation in the gene of interest. There is a need to validate NGS technologies for utilization in clinical practice.[24] Massively parallel sequencing showed 100% sensitivity in the identification of EGFR and KRAS mutations compared with 67% and 89% sensitivity shown with Sanger sequencing and pyrosequencing techniques, respectively.[23],[26]

 » Targeted Methods Top

Targeted methods can be PCR-based or non-PCR-based methods.[23] They are highly sensitive, require limited amount (5%–10%) of tumor material, and can detect known hotspot mutations in specific genes.[24] Amplification refractory mutation system (ARMS) is more sensitive than sequencing techniques in the detection of somatic mutations such as EGFR in FFPE tumor DNA samples.[27] The advantages with PCR-based assays over direct sequencing include higher sensitivity, ease of scoring, and rapid identification of responders to TKI therapy. It eliminates the need for sequencing in patients with lung adenocarcinoma.[28] Non-PCR-based methods such as SMart amplification process can detect a mutation within 30 min under isothermal conditions and in a single step.[23],[29]

The factors affecting the choice of testing method include:[23]

  • Nature of sample – High or low tumor content
  • Detection of all mutations or only specific activating mutations
  • Availability of laboratory expertise and equipment.

 » Challenges With Tissue-Based Genotyping Top

Sampling challenges

There are several barriers to acquisition of tumor tissue for sampling. The lower tumor cellularity in primary lung tumors, inaccessibility of target tumor tissue, and nature of tissue specimens obtained in patients with advanced NSCLC are some of the challenges faced in the analysis of these samples. In majority of patients, the samples are obtained by computed tomography-guided percutaneous biopsy or ultrasonography-guided endoscopic biopsy rather than by surgical resection or open thoracotomy. This affects the quantity and quality of the samples obtained for analysis. The success of molecular analysis depends on the method used for testing, absolute number of tumor cells, and the proportion of tumor cells compared with the total nucleated cells.[30] With respect to sample preservation, cross-linking of DNA can occur in FFPE samples that can result in samples being inadequate for molecular testing.[31] False-positive results can occur with preservation methods such as formalin fixation since they can show high levels of C > T/G > A transitions in the 1%–25% allele frequency range.[32]

From the patients' perspective, biopsies are uncomfortable due to invasive nature of the procedure, inconvenience, possible association with complications, and a consequent increase in health-care costs.[31]

 » Tumor Heterogeneity and Issues in Tumor Re-Biopsy Top

NSCLC tumors display both spatial and temporal heterogeneity which adds to the challenge of analysis of the tissue biopsy specimens. The highly dynamic nature of the tumor, intra- and inter-tumor heterogeneity, and limited accessibility of tumor tissue hinder the acquisition of complete genomic picture of the tumor.[32] The tumor heterogeneity interferes with the evaluation of mechanisms of resistance to TKIs when re-biopsy is done in patients with disease progression.[33] Accurate representation of all the resistance mechanisms present in tumor is not possible with a single sample. Single sample of tumor tissue also cannot identify the breadth of subclonal driver events present across multiple disease sites.[30] In patients with metastatic disease who generally have poor performance status, it is difficult to obtain tissue samples from multiple sites for testing.[33]

In patients with disease progression despite treatment, sampling bias can occur due to interspersed necrotic tissue and viable tissue. Obtaining viable tissue and sufficient amount of tissue is difficult in these patients. The procedure may also be associated with an increased risk of complications such as hemorrhage or pneumothorax.[17] Chouaid et al. assessed the feasibility and clinical utility of re-biopsy in patients with advanced NSCLC and disease progression after first-line targeted therapy. In 18% of patients, re-biopsy could not be performed. Among the samples that were histologically analyzed, 18.3% and 7.3% of the samples contained no and negligible tumor cells for molecular testing, respectively. Complications were found to be infrequent among patients who underwent the procedure.[34]

 » Liquid Biopsy Top

The highly dynamic nature of tumor with clonal evolution over time warrants longitudinal surveillance of molecular landscape of the tumor. This cannot be efficiently achieved with tissue biopsy samples and requires an alternative approach. Due to the rapid turnover rate of tumors, tumor materials such as nucleic acids, vesicles, and viable cells are constantly released into the circulation. These can be used to assess the dynamic molecular landscape of tumor by an approach referred to as liquid biopsy.[35] Liquid biopsy can be used for diagnostic purposes as well as for monitoring treatment response and evaluation of resistance mechanisms. The biomarkers of liquid biopsy include circulating cell-free tumor DNA (ctDNA), circulating tumor cells (CTCs), exosomes, tumor-derived RNA, and tumor-educated platelets.[36] Apart from circulation, they can be obtained from other biological fluids such as urine, saliva, pleural effusion, and ascites.[37]

 » Circulating Cell-Free Tumor DNA Top

The release of tumor DNA fragments into the circulation occurs due to high levels of necrosis within tumor masses and apoptosis of cancer cells.[35] ctDNA, a fraction of circulating free DNA, can constitute from <1% to >30% of the total DNA. These biomarkers carry the same genetic alterations as the tumor and these alterations can be identified and quantified.[36] Plasma samples are superior to serum samples for ctDNA analysis.[35]

Targeted and untargeted approaches are available for the analysis of ctDNA. The allelic frequency of a particular mutation and genetic alterations in specified genomic regions of plasma DNA are identified using targeted approaches. Untargeted approaches perform comprehensive analysis of tumor genome and do not require prior knowledge of the region of interest that is needed to identify specific mutations.[38] Real-time quantitative PCR (qPCR), digital PCR (dPCR), beads, emulsion, amplification, and magnetics (BEAMing), personalized analysis of rearranged ends, and deep sequencing are some of the targeted methods for ctDNA analysis.[36],[38] Untargeted approaches include whole-genome or whole-exome sequencing.[38]

In a meta-analysis of twenty studies conducted to evaluate the diagnostic performance of circulating free DNA for the detection of EGFR mutations in NSCLC, circulating free DNA was found to have adequate diagnostic accuracy in the identification of these mutations.[39] In another meta-analysis of 27 studies, the utilization of ctDNA for EGFR mutation detection in NSCLC patients was associated with 96% specificity, 62% sensitivity, and had high diagnostic accuracy. Hence, it can be considered as a primary screening test in patients with NSCLC.[40]

 » Circulating Tumor Cells Top

CTCs are released into circulation from primary tumor either by passive shedding or by intravasation and also from metastatic deposits.[35] The concentration of CTCs is low in circulation with as few as one CTC per 106–107 peripheral blood mononuclear cells in patients with advanced disease.[36] CTCs can be isolated by positive or negative enrichment based on biological properties such as expression of protein markers on the surface of the cell. The methods of isolation based on physical properties include filtration-based system, microchip, centrifugation on a Ficoll density gradient, dielectrophoresis, and spiral CTC chip.[41] The prognostic utility of CTCs has been demonstrated by Krebs et al. in patients with NSCLC. A significant predictor of worse prognosis was the number of CTCs with worse prognosis in those patients having ≥5 CTCs compared with those having <5 CTCs.[42]

The techniques for analysis of CTCs include whole-exome sequencing, whole-genome sequencing, microfluidics, arrays, and in situ RNA fluorescence in situ hybridization (FISH).[38] EGFR mutation was detected in 84% of CTC samples analyzed using ultra-deep NGS as reported by Marchetti et al. This technology can be useful in detecting mutations involved in acquired resistance to TKIs.[43] In a study comparing identification of T790M mutation in tissue biopsy and blood-based genotyping using CTC and ctDNA, the blood-based genotyping assays together detected the mutation in 35% of patients in whom tissue biopsy was negative or indeterminate.[44]

 » Exosomes Top

Exosomes are extracellular vesicles that are exuded by exocytosis that transfers information to target cell.[35],[36] They play an important role in tumor initiation and growth, metastasis, disease progression, and drug resistance.[36] They contain protein, DNA, messenger RNA (mRNA), microRNA (miRNA), and can serve as potential cancer biomarkers. In patients with limited amount of ctDNA, mRNA from exosomes is useful for analysis of cancer genome. There will be thousands of copies of mRNA per cancer cell which will be released into circulation either as circulating free RNA or in exosomes.[35] Exosomes can serve as screening and diagnostic tools in lung cancer.[45]

Circulating RNA profiling is highly valuable in patients with cancers. Somatic mutations in DNA do not reveal the genomic landscape of tumor in entirety such as epigenetic alterations or effects of miRNAs.[35] Evaluation of specific miRNA expression levels in body fluids can aid in cancer screening, staging, and response to treatment. There is a lack of consistency and standardization in methodologies used for assessing circulating miRNAs.[46]

 » Tumor-Educated Platelets Top

Tumor-educated platelets are a suitable material for blood-based genotyping. These platelets are educated through transfer of tumor-associated biomolecules.[36] Tumor-educated platelets offer a valuable platform for pan-cancer, multiclass cancer, and companion diagnostics.[47] [Table 1] enlists the ongoing trials for mutation detection in NSCLC using liquid biopsy.[48]
Table 1: Ongoing trials for mutation detection in non-small cell lung cancer using liquid biopsy

Click here to view

 » Clinical Applications of Liquid Biopsy Top

In lung cancer, liquid biopsy using CTCs may complement solid biopsy to inform effective targeted therapies. CTC profiling can predict disease progression and identify secondary mechanisms of resistance emerging in the tumor. The role of CTC as a biomarker for early diagnosis of cancer is limited due to the low levels of CTC present in circulation in the early stages of disease. Highly sensitive technologies need to be developed that can aid in the early detection of cancer.[49] Levels of circulating free DNA can be used to assess tumor burden. The estimation of levels of circulating free DNA combined with ctDNA analysis for somatic mutations serves as a valuable diagnostic tool in cancer. Early detection of cancer by liquid biopsy enables treatment at an earlier stage. Detection of promoter methylation in ctDNA can help to predict response to chemotherapy. ctDNA analysis can be used to monitor minimal residual disease and to de-escalate treatment in patients with negative minimal residual disease. It might be helpful to identify patients at an increased risk of relapse by measuring minimal residual disease so that rescue treatment can be offered to such patients to prevent relapse.[35]

Liquid biopsies can be used to monitor response to treatment and to evaluate mechanisms of resistance to systemic therapy.[35] The outcome of EGFR-TKI therapy can be effectively predicted by dynamic plasma EGFR mutation status. The inability to clear plasma EGFR mutation post-TKI therapy was associated with lower disease control, shorter progression-free survival (PFS), and shorter overall survival, and thus is an independent predictor of outcome. The assessment of plasma EGFR mutation status post-TKI therapy can help to identify patients with primary resistance and patients with disease progression despite initial response to EGFR-TKI therapy.[50] Zheng et al. detected T790M mutation in ctDNA samples in 47% of patients with progressive disease on initial treatment with a TKI. About 50% of patients with T790M mutation were detected at a median time of 2.2 months prior to clinical progressive disease. The presence of plasma T790M mutation in patients on TKI therapy was associated with poor prognosis and reflects tumor burden and metastasis.[51]

Some of the novel technologies that are emerging in the field of liquid biopsy include molecular barcode approach to improve detection of low-frequency mutant alleles,[52] plasma bisulfite sequencing,[53] nucleosome mapping,[54] and single-stranded DNA library preparation.[55]

 » Requirement of Sensitive Method for Mutation Detection in Plasma Top

ctDNA is present in blood at low concentrations along with predominant wild-type DNA originating from leukocytes. Hence, sensitive mutation detection methods are required that can identify mutant alleles present in the concentrations of <1% of the total DNA.[56],[57] It is preferable to have a mutation detection method that has high specificity over sensitivity since highly sensitive assays can lead to false-positive results.[56] The sensitivity of available techniques to analyze mutations of ctDNA varies from 0.01%–15%. Majority of the techniques can detect genetic alterations with sensitivity of at least 2%.[57]

The following points should be considered to enable successful detection of EGFR mutations in ctDNA of patients with advanced NSCLC. Plasma is preferable over serum for EGFR mutation analysis of ctDNA. Processing of blood sample to obtain plasma should be done within 4 h of collection, and plasma should be obtained by centrifugation of the blood sample. A clean sample can be obtained by a second, high-speed spin (before or after freeze/thaw 3000–16,000 g) in a microcentrifuge. If processing can be done only at a later time, then the use of stabilization collection tubes containing fixatives should be considered. Plasma should be stored at −20°C or −80°C. DNA extraction methods specific for ctDNA should be used since it is fragmented and present in low concentrations. PCR-based methods that increase the proportion of mutant to wild-type DNA should be preferred over traditional methods such as Sanger and pyrosequencing. Droplet dPCR (ddPCR) and BEAMing show greater sensitivity than that of traditional PCR methods. Quantification of mutant EGFR can be done using methods such as ddPCR and NGS that can be used to assess progression of disease and response to treatment. Reliable and efficient methods for diagnostic use include denaturing high-pressure liquid chromatography (HPLC), mass spectrometry genotyping, and HRM analysis.[56]

 » Plasma Genotyping Platforms Top

The genotyping platforms for liquid biopsy include ddPCR, peptide nucleic acid-mediated PCR, cobas® EGFR mutation test, NGS, Scorpion ARMS, BEAMing, and HPLC.[58] The cobas® EGFR mutation test version 2 approved by the Food and Drug Administration in 2015 is designed to detect the following EGFR mutations: deletion mutation in exon 19; T790M and S7681 substitution mutations and insertion mutations in exon 20; G719X substitution mutation in exon 18; and L858R and L861Q substitution mutations in exon 21.[59],[60] The specificity of these validated assays comparing circulating free DNA to tissue biopsy ranges from 90% to 100%.[61] These assays show variable sensitivity (30%–85%), and the turnaround time also varies across these platforms. The turnaround time for available NGS platforms is around 2 weeks. [Table 2] shows the sensitivity, specificity, and concordance of various platforms used for the analysis of circulating free DNA with tumor biopsy as obtained from various trials.[62],[63],[64],[65],[66],[67],[68],[69],[70],[71]
Table 2: Performance characteristics of various platforms for circulating free DNA analysis relative to tumor biopsy

Click here to view

In a meta-analysis of 27 studies for the detection of EGFR mutations in circulating free DNA in NSCLC patients, the pooled sensitivity and specificity of various platforms were 0.60 and 0.94, respectively. The various platforms used in these studies were ARMS, mutant-enriched PCR, denaturing HPLC, HRM analysis, peptide nucleic acid-mediated PCR clamping, peptide nucleic acid-locked nucleic acid PCR, allele-specific PCR, dPCR, mutant-enriched PCR, mutant-enriched liquid chip, NGS, and deep sequencing.[72]

The utility of CTCs for detection of genetic alteration in routine clinical practice is lesser than that of circulating free DNA.[60]

 » Droplet Digital Polymerase Chain Reaction Top

Zhu et al. compared demonstrated sufficient concordance of ddPCR in the detection of EGFR mutations in NSCLC patients with tissue samples taken from tumor. The sensitivity and specificity of ddPCR in the detection of mutant L858R mutations were 76.19% and 96.55%, respectively, compared with tissue samples. For exon 19 deletion, the sensitivity and specificity were 88.89% and 100%, respectively. A significant positive correlation was also established between baseline concentrations of plasma-mutant EGFR, exon 19 deletions, and response to TKI therapy.[73] In patients with EGFR-TKI resistance, the concordance of ddPCR for detection of EGFR-sensitizing mutations in plasma was 78% compared with tissue or malignant fluid samples. The concordance was 65.9% for the detection of T790M mutation compared with tissue or malignant fluid samples. The sensitivity and specificity of ddPCR for detection of T790M mutation were 64.5% and 70%, respectively.[74]

 » Amplification Refractory Mutation System-Polymerase Chain Reaction Top

Liu et al. compared direct sequencing with ARMS for EGFR mutation analysis in the body fluids (plasma, pleural fluid) of patients who have undergone TKI therapy. Among patients who provided plasma, ARMS assay was able to detect mutation-positive patients, whereas direct sequencing failed to detect mutation-positive patients. Although positive mutation results are good indicators of TKI treatment, there was a poor correlation between negative results and response to TKI therapy. This can be attributed to false-negative results which can be minimized using more sensitive techniques than ARMS such as dPCR.[75]

Scorpion ARMS PCR is relatively cheap and has higher sensitivity than qPCR. This method can identify only those genetic alterations which its probes are designed to detect.[76] Using Scorpion ARMS method for EGFR mutation detection in 94 patients with NSCLC, the detection rate, sensitivity, and specificity of using plasma samples were 20%, 50%, and 100%, respectively, compared with tissue samples. The consistency of EGFR mutation status between plasma and tissue samples was 81%, and the overall concordance was 80% between plasma and tissue samples.[77]

 » Beads, Emulsion, Amplification, and Magnetics Top

BEAMing can detect genetic alterations even when present in at low concentrations.[57] This standardized and extensively validated method is reliable and has a short turnaround time of 5–10 days. Karlovich et al. assessed concordance between BEAMing plasma test and tumor tissue test for EGFR mutation status in 77 patients with NSCLC who had received EGFR-TKI therapy at least once. The positive percentage agreement (identification of the same mutation in plasma samples and tissue samples) between BEAMing plasma test and cobas ® tumor test for activating mutations and T790M mutations was 82% and 73%, respectively. The BEAMing plasma test was able to detect more patients with T790M mutation than that of the tumor test. The objective response rate to rociletinib in T790M-positive patients identified by BEAMing was comparable to patients detected by tumor test.[78]

In patients with advanced cancers, EGFR mutational analysis using BEAMing technology in plasma circulating free DNA and archival tissue mutational analysis was concordant in 99% of cases.[79] Apart from its high sensitivity, BEAMing can enable quantification of mutant allele and estimate the fraction of T790M mutation in tumor cells. This can aid in the monitoring of disease progression.[80]

 » Next-Generation Sequencing Top

NGS technologies allow sequencing of large portions of genome and enable detection of multiple mutations in multiple genes. Very deep coverage of clinically relevant targets is possible by focusing only on limited sequences of frequently mutated genes.[57],[81] NGS technologies can detect ctDNA present in low concentrations in plasma or body fluids.[81] Deep sequencing can be used to monitor the course of disease by performing repeated assays at multiple time points in the patient.[82] Though NGS is an efficient method, it lacks required sensitivity.[36] The various approaches in NGS include whole-genome, whole-exome, transcriptome, and targeted sequencing approaches.[83]

Uchida et al. used deep sequencing of plasma DNA to detect mutations in 288 patients with NSCLC and compared the results with genotyping results from tissue samples. The sensitivity and specificity for exon 19 deletions were 50.9% and 98.0%, respectively. The sensitivity and specificity for L858R mutations were 51.9% and 94.1%, respectively. Thus, high specificity observed with deep sequencing indicates that TKI therapy can be recommended in patients with positive results. This technique of mutation detection should be used in advanced stages of NSCLC. Patients with lesions for which biopsy sampling is difficult, with uncertain biopsy results, or those who do not want to undergo the biopsy procedure, are ideal candidates for deep sequencing.[83]

 » Plasma Genotyping for T790m Mutation Top

The determination of T790M mutation is of prime importance due to the proven efficacy of third-generation TKIs in patients harboring this mutation. Repeated tissue biopsies to ascertain the presence of this predictive biomarker may be hindered due to inaccessibility of tumor or lack of patient preference for biopsy. Plasma genotyping can be a suitable option in such situations.[17] In patients with acquired EGFR-TKI resistance and evidence of common sensitizing EGFR mutation, the sensitivity of plasma genotyping using BEAMing method was estimated as 70% by Oxnard et al. This method was able to detect T790M mutation in the plasma of 31% patients with T790M-negative tumors. Patients presenting with acquired resistance should undergo plasma genotyping for T790M mutation initially rather than tumor biopsy. If the test is positive, tumor biopsy can be avoided. However, if it is negative, tumor biopsy is warranted to investigate T790M mutation in tumor tissue since false results can occur with plasma genotyping.[84]

Among 260 patients with acquired EGFR-TKI resistance, ddPCR detected T790M mutation in the plasma of 70 patients (28.8%) and EGFR-activating mutations in 120 (46.2%) patients as reported by Takahama et al. ddPCR assay is a suitable method for the identification of plasma T790M mutations and in patients with inaccessible tumors for re-biopsy.[74] The presence of T790M mutation in plasma indicates high tumor burden and is associated with reduced PFS compared with T790M mutation only in tumor (16.5 months vs. 9.3 months).[84],[85]

 » Plasma Genotyping Assay Validation in Lung Cancer Top

The prospectively validated assays include denaturing HPLC, ddPCR, and NGS. The retrospectively validated assays include BEAMing, cobas® EGFR mutation test, NGS, Scorpion ARMS, peptide nucleic acid-mediated PCR, dPCR, HRM analysis, and mass spectrometry genotyping.[86]

 » Cross-Platform Comparisons Top

Thress et al. compared two digital platforms and two nondigital platforms for mutational analysis of ctDNA in plasma samples of advanced NSCLC patients. For EGFR-sensitizing mutations, sensitivity and specificity were high across nondigital (cobas® and Therascreen ) and digital platforms (ddPCR and BEAMing digital PCR). For plasma T790M mutation, the performance of digital platforms was better than the nondigital platforms. The sensitivity and specificity were 73% and 67%, respectively, for cobas ® test, whereas they were 81% and 58%, respectively, for BEAMing dPCR.[87]

In patients with acquired EGFR-TKI resistance, the T790M mutation detection rate was higher with ddPCR assay than ARMS assay (46.7% vs. 25.3%), demonstrating higher sensitivity of ddPCR assay.[88] Xu et al. compared four platforms to detect EGFR mutations in ctDNA samples of NSCLC patients. The detection rate of ADx-ARMS was lower than firefly NGS, ddPCR, cobas ®-ARMS methods demonstrating superior sensitivity with these three methods than ADx-ARMS. The latter is more suitable for qualitative detection of EGFR mutations, with allele frequency >1% in plasma and tissue samples.[89]

qPCR as well as ddPCR can be used for quantitative estimation of circulating miRNAs which have been identified as biomarkers of lung cancer, with ddPCR assay showing similar or greater precision.[90]

 » Challenges With Liquid Biopsy Top

Though liquid biopsy looks promising in the field of lung cancer, it does come with some limitations. A tissue biopsy will be required to establish the histological subtype of lung cancer.[33] Liquid biopsy can be challenging for early detection of cancer due to low abundance of tumor materials such as CTCs.[49] The presence of mutations in plasma does not establish that these mutations are coming from presumed lung cancer, and the absence of mutations in genes such as EGFR-coding gene or KRAS fails to establish the histological subtype of lung cancer. In EGFR-TKI-resistant cases, liquid biopsy technologies cannot detect histological transformation of tumor to small-cell cancer or epithelial–mesenchymal transition.[33],[91] The analysis of ctDNA can yield false-negative results and only address genetic alterations and not the tumor status at DNA, RNA, and protein levels. ctDNA reflects the genome of dying tumor cells and not the existing tumor cells or resistant subpopulations. It cannot provide information regarding the exact origin of mutation.[92]

The underlying biology of the release of both CTC and ctDNA from tumor cells is still not clearly understood. This is essential to understand whether plasma genotyping truly represents the physiological nature of disease and is a reliable method to detect and monitor disease. It is poorly understood whether the tumor-derived materials are released from metastatic sites to circulation or are released in equal amounts by all tumor cells.[38] In contrast with CTCs, more studies exist regarding the clinical utility of ctDNA in lung cancer. However, it is not possible to perform in situ and morphological analyses using immunocytochemistry and FISH using ctDNA. Randomized controlled trials to establish the role of liquid biopsy in guiding treatment strategy or its role in complementing tissue biopsy are limited.[91]

In order to make liquid biopsy assays reliable, it is necessary to standardize the preanalytical steps, validate the analytical steps, and establish the clinical relevance to evaluate ctDNA at different time points during the course of the disease. The performance characteristics of these assays must be aligned with the recommendations of regulatory agencies to ensure standardization and reproducibility.[38] Various diverse platforms for ctDNA analysis should be optimized and standardized.

 » Conclusion Top

In NSCLC patients, postprogression, re-biopsy, and detection of EGFR T790M mutation remain essential for therapy selection. Re-biopsy and detection of EGFR T790M mutation can be done in gold standard tissue genotyping and minimally invasive plasma genotyping. The minimally invasive plasma genotyping is a promising approach and complementary to conventional tissue genotyping. However, the plasma genotyping assays and platforms need to be standardized and validated to provide reliable, reproducible, and robust results. With the emergence of T790M mutation, noninvasive genotyping for this mutation offers an approach that may be broadly implemented rather than the conventional tissue re-biopsy and testing. There is a need for more studies on clinical validation of plasma genotyping to become a routine practice in clinics.[93]


The authors acknowledge AstraZeneca Pharma India Ltd and Indegene Lifesciences 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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