1. Introduction
Lung cancer is the leading cause of cancer-related death around the world. Nearly 85% of lung cancer patients have non–small cell lung cancer (NSCLC), with an overall 5-year survival rate of less than 15% [
1,
2]. Epidermal growth factor receptor (EGFR) mutations are the most frequent oncogenic driver mutations found in NSCLC. Mutations of the
EGFR gene that occur in exons 18 to 21 within the kinase domain, leading to the activation of the kinase activity by a ligand-independent mechanism, are referred to as “activating mutations”.
EGFR-activating mutations are highly associated with tumor responses to EGFR-TKIs.
EGFR exon 19 deletions and L858R mutation are the two most common activating mutations in NSCLC [
3]. More than 50% of Asian NSCLC patients were found to harbor
EGFR-activating mutations, which are more prevalent in female and never-smokers [
4]. EGFR tyrosine kinase inhibitors (EGFR-TKIs) treatment, demonstrating superior progression-free survival, higher tumor response rates and a better quality of life, are now recommended as the primary therapy for advanced-stage NSCLC patients with
EGFR-activating mutations [
5,
6,
7,
8,
9]. However, most patients who initially respond to first- or second-generation EGFR-TKIs will eventually undergo disease progression after a median of 9 to 14 months due to acquired resistance [
6,
7,
8,
10,
11]. Approximately 50 to 60% of cases of acquired resistance for first- and second-generation EGFR-TKIs are due to the emergence of secondary
EGFR T790M mutation that affects the “gatekeeper residue” in the catalytic domain of the kinase, leading to steric hindrance of EGFR-TKI binding due to the presence of the bulkier methionine side chain in the ATP-kinase-binding pocket and subsequently abolishing the potency of ATP-competitive TKIs [
3,
12]. Recently, the third-generation EGFR-TKI, osimertinib, targeting both
EGFR-activating mutations as well as the gatekeeper
EGFR T790M mutation, has been approved as a first-line treatment for patients with EGFR-mutant metastatic NSCLC and as a second-line therapy for patients who have developed
EGFR T790M mutation after failure of the first- and/or second-generation EGFR-TKI treatment [
13,
14]. Therefore, the continuous assessment of tumor
EGFR mutation status during the course of disease is necessary for the better management of NSCLC patients, particularly for the early identification of the resistance mechanisms.
Currently, the gold standard for assessing
EGFR mutations is to analyze the tumor tissues. However, obtaining tumor samples through tumor biopsies has certain limitations [
15,
16]. First, tumor biopsy is an invasive and potentially risky procedure. Second, a small amount of tumor tissue obtained from a single-site biopsy has a high failure rate in molecular testing and is further affected by intra-tumoral heterogeneity and sampling bias [
17]. Recently, testing plasma circulating cell-free DNA (cfDNA), commonly known as liquid biopsy, has been highlighted as a promising alternative. Unlike traditional tumor biopsy, liquid biopsy is less invasive and allows for frequent sampling during follow-up. Liquid biopsy contains tumor DNA released from both the primary and/or metastatic tumor sites, which can better reflect the molecular heterogeneity of tumors [
18,
19].
Given that the tumor tissue is extremely heterogeneous, and the concentration of tumor DNA in peripheral blood is extremely low and varies between patients, molecular methods with a high detection sensitivity are required for more accurate diagnostics. The cobas EGFR Mutation Test is a real-time polymerase chain reaction (PCR) test that can qualitatively identify 42 mutations in exons 18, 19, 20 and 21 of the
EGFR gene, including the T790M resistance mutation, by using allele-specific PCR primers specifically amplifying the targeted mutant sequences rather than wild-type sequences and/or other human genomic DNA. It has been clinically validated as a companion diagnostic (CDx) for EGFR-TKIs therapy in patients with advanced NSCLC with a detection sensitivity of around 5% for tissue-derived DNA [
20]. Droplet digital PCR (ddPCR) is an ultra-sensitive assay combining microfluidics technology with TaqMan-based quantitative PCR (qPCR) to measure absolute mutation alleles through clonal amplification and fluorescence detection of tens of thousands of individual template molecules in a single reaction. Unlike classic qPCR, which depends on calibration curves for target sequence quantification, ddPCR collects fluorescence signals via end-point measurements and utilizes Poisson statistics to calculate the target concentrations, which could avoid the pitfalls associated with the variations in reaction efficiencies. Therefore, ddPCR has emerged as a promising tool for the detection and absolute quantification of gene mutations below 1% [
21,
22,
23,
24]. As ddPCR is increasingly implemented in clinical practice for detecting somatic mutations that are present at low frequencies in tumors or circulating cell-free DNA, the current study aimed to evaluate the clinical utility of assessing both plasma and tissue
EGFR mutations using ultra-sensitive ddPCR assays prior to the treatment and at disease progression in monitoring the efficacies and outcomes of NSCLC patients treated with EGFR-TKIs.
3. Discussion
Accurately identifying tumors that harbor
EGFR-activating as well as T790M resistance mutations is critical for the precision management of NSCLC [
26]. In this study, through the analyses of
EGFR mutation status using ddPCR performed in paired tumor and plasma samples obtained from 137 patients with advanced NSCLC prior to their EGFR-TKI treatment and at disease progression, we demonstrated that (1) baseline plasma
EGFR-activating mutation status can be used as a predictive marker for EGFR-TKI therapy; (2)
EGFR T790M mutation pre-existing as a minor subpopulation prior to EGFR-TKI treatment is not associated with the emergence of acquired resistance at disease progression; (3) Tissue testing for
EGFR T790M mutation at disease progression cannot be used as a stand-alone assay, and the analysis of plasma should also be used to more precisely identify patients who might benefit from the subsequent third-generation EGFR-TKI treatment.
By using ddPCR, we found that plasma
EGFR-activating mutations were detected in 65% (89/137) of patients prior to EGFR-TKI treatment. The positivity of
EGFR-activating mutation in baseline plasma has been addressed in several previous studies, with a range from 20% to 73% [
27]. The variability among studies may result from the differences in the assay sensitivity and disease stages of the enrolled patients. Our results showed that patients with detectable baseline plasma
EGFR-activating mutations had a shorter PFS and OS compared to those without, suggesting that the absence of detectable
EGFR-activating mutations in baseline plasma might be used as an indicator for low distant spreading activities and low systemic tumor burden in NSCLC. Our observation of an improved PFS and/or OS to EGFR-TKIs treatment in patients without
EGFR-activating mutations detected in baseline plasma is also consistent with the previous findings [
28,
29,
30].
Baseline plasma
EGFR mutation testing is now recommended in the College of American Pathologists (CAP)/International Association for the Study of Lung Cancer (IASLC)/Association for Molecular Pathology (AMP) guidelines for the molecular testing of patients with NSCLC as an alternative for a diagnostic tissue biopsy in cases with insufficient tumor tissue specimens or where tissue specimens are not obtainable; however, its prognostic value for predicting EGFR-TKI outcomes, which has been demonstrated in previous studies, has not yet been applied to clinical practice [
31]. One major obstacle for translating plasma
EGFR mutation testing into routine clinical practice is the lack of standardization of methods for assessing tumor mutations. Therefore, the further evaluation of larger groups of NSCLC patients in prospective studies with respect to diagnosis and prognosis may be warranted for the wide implementation of liquid biopsy.
The seminal mechanism of acquired resistance to first- or second-generation EGFR-TKIs is known to be the emergence of
EGFR T790M mutation. It is not clear whether
EGFR T790M mutation in NSCLC patients who have relapsed from first- or second-generation EGFR-TKIs treatment is acquired during disease progression or develops from the pre-existing
EGFR T790M clones in treatment-naïve patients as a minor population. The presence of pre-treatment
EGFR T790M mutation in NSCLC has been discovered in several studies using highly sensitive detection methods, such as mass spectrometry, the scorpion amplification refractory mutation system, colony hybridization assays, etc.; the reported prevalence ranges from 20 to 80% [
32,
33,
34,
35].
In the present study,
EGFR T790M mutation in 20.4% (28/137) of the EGFR-mutated, treatment-naïve NSCLC tumors was only detected by using the ddPCR platform but not the cobas EGFR mutation Test, and the FA of
EGFR T790M is lower than that of
EGFR-activating mutations in baseline tissue and plasma samples, showing that
EGFR T790M pre-existing as a minor subpopulation in treatment-naïve, EGFR-mutated NSCLC has undergone clonal expansion in response to the selection pressure by the first- or second-generation EGFR-TKIs treatment. However, through analyses of
EGFR mutations in plasma and tissue at baseline and disease progression, we found that the presence of
EGFR T790M mutation at baseline was not statistically associated with the emergence of acquired
EGFR T790M resistance at disease progression. One possible explanation is that the intratumoral heterogeneity of
EGFR T790M mutation in tumor tissues results in sampling bias during the tumor biopsy procedures, which contributes to underestimating the incidence of this mutation and leads to the lack of statistical differences found in the present study. Indeed, a study evaluating
EGFR T790M mutation in the sequential rebiopsies, along with the course of first-generation EGFR-TKI treatment, found that some patients who were
EGFR T790M-positive at the first post-TKI biopsy became
EGFR T790M-negative in later post-TKI rebiopsies, and vice versa, which is suggestive of the intratumoral heterogeneity of the mutation [
36]. Moreover, the involvement of other molecular changes in the resistance mechanisms may also contribute to the results. In the present study, the concordance rate for
EGFR T790M mutation was only 71.4% (25/35) between paired tissue and plasma samples at disease progression, with some being
EGFR T790M-positive in the plasma and negative in the tissue, and vice versa. These findings indicate that tissue or plasma testing for
EGFR T790M mutation should not be used as a stand-alone assay for selecting patients that are eligible for the subsequent osimertinib treatment, and the repeat or combined testing should be considered.
Our findings that pre-treatment
EGFR T790M mutation was significantly associated with brain metastasis in patients with EGFR-mutated tumors receiving first- or second-generation EGFR-TKIs suggested that therapeutically targeting the pre-existing minor subpopulation harboring T790M mutation may have the advantage of preventing the development of brain metastasis in NSCLC. In the FLAURA trial, an analysis of a subset of treatment-naïve patients with EGFR-mutated advanced NSCLC and CNS metastases showed that the PFS was longer for patients receiving osimertinib compared to those receiving either gefitinib or erlotinib (15.2 versus 9.6 months; HR 0.47, 95% CI 0.30–0.74), indicating that osimertinib has a higher CNS efficacy in patients with untreated EGFR-mutated NSCLC [
14]. Ballard et al. has further validated the CNS activity of osimertinib in a mouse model [
37]. The findings in our current study of an association between pre-treatment
EGFR T790M and brain metastasis might also support the use of osimertinib as a front-line EGFR-TKI treatment in preventing CNS progression.
The prognostic value of pre-treatment
EGFR T790M mutation in advanced NSCLC patients treated with TKIs remains inconclusive. Some studies showed that patients with detectable
EGFR T790M mutation had a shorter PFS, but this had no impact on OS, while others found that the presence of pre-treatment
EGFR T790M mutation indicated favorable outcomes in advanced NSCLC patients treated with EGFR-TKIs [
32,
33,
34,
35]. In our study, we found no significant difference in patient survival between those with and without pre-treatment
EGFR T790M. Possible explanations for the discordance include the differences in assay methodologies, with different sensitivities used among studies, the source of tumor samples (e.g., surgical resection or biopsies) used for analysis and the first- or second-generation EGFR TKIs chosen for treatment. Noteworthily, most patients enrolled in other studies were treated with first-generation EGFR-TKIs, such as gefitinib or erlotinib, but, in our present study, approximately 50% of patients were treated with the second-generation EGFR-TKI afatinib as the first-line therapy. In the phase IIB LUX-Lung 7 trial comparing afatinib and gefitinib as the first-line treatment for EGFR-mutated NSCLC patients, the researchers demonstrated that afatinib significantly improved PFS and time-to-treatment failure compared with gefitinib [
38]. In addition, afatinib has been shown to inhibit the growth of gefitinib-resistant lung cancer cells harboring low levels of
EGFR T790M mutation, but not those with high levels of
EGFR T790M mutation [
25]. Indeed, in this study, we did find that the frequency of developing acquired
EGFR T790M mutation in patients treated with gefitinib or erlotinib was higher than those treated with afatinib (32.6% vs. 19.4%). In addition, our results showed that patients with detectable baseline
EGFR-activating mutations are significantly associated with bone metastasis. Previous studies have found that the incidence of
EGFR-activating mutations in bone metastasis is higher than that in primary adenocarcinomas or metastases to other organs. A study by Furugaki et al. reported that the inhibition of EGFR signaling by erlotinib prevents the tumor-induced osteolytic invasion of NCI-H292 cell lines [
39]. These findings suggest that the activation of EGFR signaling promotes the osteolytic invasion and metastasis of tumor cells and provides supportive evidence to our result that baseline plasma
EGFR-activating mutation status might serve as a predictive marker for the development of bone metastasis in NSCLC.
There were limitations in our current study. First, it was a single-center study with a small sample size due to the difficulty in longitudinally collecting the tissue and plasma samples in advanced NSCLC patients prior to treatment and at disease progression, and further investigation in a larger cohort may be required to confirm the findings. Second, the assay sensitivity and specificity towards EGFR mutation testing using the ddPCR platform cannot be accurately evaluated in this study because of a lack of enrolled EGFR mutation-negative patients. Third, patients enrolled in this study were at advanced stages, and their tumor tissues were obtained via tumor biopsy. The intratumor heterogeneity of EGFR T790M mutation may lead to an underestimation of the prevalence due to sampling bias, which may, in turn, influence its clinical impact.
In conclusion, our study demonstrated the significance of using the ultra-sensitive ddPCR platform for the dynamic assessment of tissue and plasma EGFR mutation status at baseline and disease progression in EGFR mutant NSCLC treated with EGFR-TKIs. In addition to evaluating the EGFR-TKI treatment efficacy and the emergence of resistance mechanisms, the use of the ddPCR method for EGFR mutation detection is proven to be valuable in predicting tumor metastasis and patient outcomes.