Background
Lung cancer is one of the leading causes of cancer related deaths worldwide [
1]. Administration of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) to patients with activating mutations in the
EGFR gene, especially exon 19 deletions and exon 21 p.L858R point mutations, has significantly improved treatment and outcome of advanced-stage lung cancer patients [
2].
Five to 50% of patients with lung adenocarcinomas carry activating mutations within the
EGFR gene with huge differences between geographical distribution and populations [
3]. Activating mutations confer patients susceptible to treatment with EGFR-tyrosine kinase inhibitors (TKIs). Objective tumour shrinkage is reported in approximately 75% of patients [
3]. Nevertheless, acquired resistance to TKIs and secondary progression is being observed after a median time of 8 to 14 months in nearly all patients [
4].
Until the emergence of osimertinib, first-line therapies were mostly administered with reversible (gefitinib, erlotinib) [
5,
6] or irreversible TKIs (afatinib) [
7]. Molecular analyses revealed a limited number of different resistance mechanisms. The most frequent mechanism (50–60%) is the gate-keeper point mutation p.T790M which lowers affinity of first-line TKIs to the ATP binding pocket [
8,
9]. Less frequent resistance mechanisms (5–15%) are the activation of bypass receptor tyrosine kinases, such as
ERBB2 and
MET amplifications [
10,
11]. Infrequently, mutations within the genes encoding the downstream signalling molecules BRAF, KRAS, PIK3CA and CTNNB1 are observed [
4]. A completely different and poorly understood mechanism abolishing sensitivity towards EGFR TKI involves the histological transformation into small cell or sarcomatoid lung cancer phenotypes [
12]. Also compound resistance by multiple mechanisms in the same or in different tumour locations have been encountered [
13].
As different resistance mechanisms require precise diagnostics and elicit a wide portfolio of different and effective second-line therapies [
14], we here addressed the question whether the frequencies of resistance mechanisms differ between first-line therapies with reversible and irreversible TKIs. So far the prevalence of the
EGFR p.T790M mutation and other resistance mechanisms after treatment with reversible first-generation EGFR TKI was investigated in different studies with low patient numbers (
n = 37) as well as larger cohorts (
n = 155) [
15]. Especially, the mutational spectrum of irreversible second-generation EGFR TKI afatinib was only investigated in studies with low patient numbers (
n = 4,
n = 20) [
16,
17]. Moreover, these studies included patients with second-line EGFR-TKI treatment. Therefore we compiled diagnostic and follow-up data of two very large German pathology centres.
Discussion
The present retrospective study confirmed that the major mechanism of resistance to afatinib treatment is the
EGFR p.T790M gatekeeper mutation. Nevertheless, the resistance mutation was detected with a lower prevalence than for reversible EGFR TKIs (erlotinib and gefitinib), 45% vs 64%
p = 0.02. This is in contrast to previous reports by Wu et al., who showed a similar prevalence for
EGFR p.T790M after afatinib or reversible EGFR TKI treatment (both 50–60%,
n = 14,first-line afatinib). Due to the relatively small patient population of first-line afatinib-treated patients with
n = 14, Wu et al. could not detect a statistically significant difference in the prevalence of EGFR p.T790M acquisition between reversible and irreversible EGFR TKI (
p = 0.83) ( [
16]). Two further prospective studies by Campo et al. and Tanaka et al. confirmed the lower prevalence of p.T790M in irreversible EGFR TKI treated patients. Campo et al. detected the EGFR p.T790M in 36% of afatinib treated patients (
n = 11) [
22]. Tanaka and co-workers deciphered the acquisition of p.T790M in 43% of afatinib treated patients (
n = 37) [
17]. Nevertheless, both studies present small cohorts of afatinib treated patients, which might be too low to draw significant conclusions from. However, our study presents the same numeric trend of a lower prevalence of p.T790M in irreversible EGFR TKI treated patients, which was determined to be statistically significant. Still,
EGFR p.T790M mutation is the most prominent mechanism of resistance in afatinib treated patients. These findings imply that afatinib-treated patients should equally benefit from treatment with third-generation
EGFR TKIs, like osimertinib, and need to be screened for emergence of the p.T790M resistance mutation. These novel emerging inhibitors are specific for the
EGFR p.T790M mutated isoform of the EGFR receptor [
23,
24]. According to El Kadi and coworkers, the formation of EGFR T790M mutation is initiated by AICDA-mediated deamination of the 5-methylcytosine following therapy with either of the EGFR TKI. Nevertheless they observed differential gene expression of AICDA under different treatment conditions (type and dose of EGFR TKI) [
25]. Therefore, a different frequency of T790M acquisition under reversible and irreversible EGFR TKI is conceivable. Moreover the rate of residual growing cancer cells under/after EGFR TKI therapy that can support AICDA- mediated deamination may differ between various TKI, thereby leading to different frequencies of acquired T790M.
Small cell lung cancer transformation has been reported as an alternative mechanism of resistance to first-generation
EGFR TKI in 3–14% of
EGFR TKI-treated patients [
15,
26]. This transformation arises upon TKI blockade of
EGFR signalling in combination with additional mutations, such as inactivation of
RB1. Within the present study, we did only detect one transformation into small cell lung cancer as resistance mechanism to afatinib therapy and none in the population of first-generation EGFR TKI treated patients. Previous reports on smaller populations did not find transformation as a resistance mechanism to afatinib therapy [
16,
17]. The detection of only one event in a cohort of 54 patients suggests that SCLC transformation is an even rarer event of resistance acquisition than in reversible
EGFR TKI treated patients. Nevertheless identification of histological transformation, especially of resistant patients, that do not show any molecular mechanism, remains to be critical for treatment recommendations.
One re-biopsy sample showed an activating
CTNNB1 mutation, which is reported to confer resistance to
EGFR therapies in non-small cell lung cancer [
27]. No other acquired resistance mutations within the
BRAF,
KRAS or
PIK3CA genes were identified in the present study. This could be either characteristic for afatinib treatment or due to the relatively small sample size. Nevertheless, two prospective studies on afatinib treated patients gathered the same observations [
17,
22]. This is in contrast to first-generation
EGFR TKI treatment, where
BRAF and
PIK3CA mutations account for 1 and 5% of resistance to treatment, respectively [
28]. A review of Westover et al. [
29] calculated KRAS mutation as a mechanism of EGFR TKI resistance to be at a frequency of approximately 1% from the majority of previously published data on resistance acquisition. Nevertheless, the underlying studies included all patients under EGFR TKI therapy without taking early progressors upon pre-existent resistance clones into account. Our study in contrast focused on acquired resistance, explicitly excluding early progressors. Only patients with a response to EGFR TKI therapy of at least 6 month were considered to be truly sensitive to therapy and did not carry any subclones with primary resistance mutations. This could be the reason for not identifying any KRAS mutation within the presented cohort of this study. Further, acquired resistance mechanisms via additional
EGFR mutations (e.g. exon 20 duplication or p.D761Y and p.L747S) were not found either. This is in contrast to previous studies on first-generation EGFR TKIs, where up to 10% of patients under
EGFR TKI therapy developed rare secondary mutations within the
EGFR gene [
30].
Activation of alternative pathways is the second common mechanism of resistance to EGFR TKIs. The emergence of anti-pan-HER treatment options (as afatinib) to block these alternative pathways by inhibiting phosphorylation of other HER family members was thought to solve this problem. Nevertheless, within this study, we examined downstream activation of the AKT pathway via amplification of the genes, encoding for
MET and
ERBB2 to be equally abundant (2% vs 3%) in specific and pan-HER TKI treated patients. Downstream activation of the AKT pathway via amplification of the gene encoding the transmembrane kinase MET was shown to be the prominent alternative mechanism of resistance to first-generation EGFR TKI with 22% of cases [
30]. Resistance acquisition by amplification of
ERBB2 is less common with a reported occurrence of 12% [
30]
. We here observed
MET and
ERBB2 amplifications as exclusive mechanisms of resistance at lower frequencies than previously described. Additionally we found those in combination with other acquired resistance mechanisms like p.T790M or as a combination of
ERBB2 and
MET amplification in the absence of a second-site mutation. The latter one is proposed to signal parallel to EGFR and thereby reactivates downstream signalling of the pathway [
27].
Since,
EGFR p.T790M is still the most common mechanism of resistance to afatinib, similar to reversible
EGFR TKI, a comparison of patients´ prognosis and form of disease upon basis of the mutational profile should be made. Previously, patients were shown to have a better prognosis and more indolent form of disease progression upon the presence of an
EGFR exon 19 deletion [
31‐
34]. Matsuo and co-workers investigated whether there was an association of the (primary)
EGFR driver mutation with the occurrence and frequency of
EGFR p.T790M and the period of response to EGFR TKI. They observed a higher occurrence of
EGFR p.T790M in patients with primary
EGFR exon 19 deletions in contrast to
EGFR p.L858R mutation (26 of 41 patients 63% vs 5 of 13 patients 38%) [
35].
These findings are comparable to the prevalences observed within our cohort of first-generation EGFR TKI treatment, where primary EGFR exon 19 mutated patients developed a p.T790M more frequently than p.L858R mutated patients (74 and 54%). Interestingly, frequencies of p.T790M acquisition under afatinib therapy are comparable between primary EGFR exon 19 mutated patients and patients with a primary EGFR p.L858R mutation (44 and 45%). Matsuo and co-workers did not separate first-generation and second-generation TKIs and had only three afatinib patients in total, while in our study 55 afatinib patients were analysed. Therefore frequencies of co-occurrence of.primary EGFR mutation and p.T790M acquisition coincide with Matsuos´ observations for first-generation EGFR TKI but differ for afatinib. In our study, a clear numeric difference in the co-occurrence of primary driver mutation and frequency of p.T790M development can be seen in reversible EGFR TKI compared to afatinib-treated patients.
Treatment duration until progress under irreversible EGFR TKI in patients with or without acquired p.T790M revealed a comparable duration of 11 and 12 months. For p.T790M mutated patients, Tanaka et al. found a comparable time to progression of 12 months. In contrast, they observed that the duration of p.T790M negative patients under afatinib therapy was much lower with 4.5 months. This strong discrepancy can be explained by the differential study setting. Tanaka et al. analysed all patients under first-line afatinib therapy, while our study excluded early progressors (< 6 months until clinical progress). We further observed a prolonged PFS of patients under first-generation EGFR TKI, but this finding cannot be compared to results of of clinical trials (e.g.LUX-Lung 7, Archer1050) as our cohort included patients with a PFS > 6 months only. The differential study design to all published data on afatinib so far, is the fact, that patients with early progression (< 6 months) were excluded from this study. The data do not show the median PFS for the different generation of TKI for all patients treated, but only for the cohort with acquired resistance. The values are not comparable to median PFS from other studies, but rather should be seen as duration under therapy until resistance acquisition.
Molecular follow-up is of primary importance for second-line treatment decisions. As we could show within this study, the gatekeeper mutation p.T790M is the most prominent mechanism of resistance among all available first and second-generation EGFR TKI. This favours the majority of relapsed patients for third-generation EGFR TKI therapy (osimertinib), thereby prolonging overall survival. Nevertheless, the differential response of early and late progressors of first and second-generation EGFR TKI patients to osimertinib should be considered. Early progressors are characterized by the pre-existence of sub-clones carrying
EGFR p.T790M that expand under EGFR TKI therapy. Those are supposed to better respond to osimertinib than late p.T790M resistant tumours that evolve from initially drug-tolerant cells [
36,
37]. In conclusion, late progressors are the target population that profits more from first-line afatinib therapy followed by third-generation EGFR (osimertinib) therapy. In contrast, early progressors are supposed to profit from first-line osimertinib therapy [
36,
38,
39]. Osimertinib, as probably the most effective way to prevent acquisition of the T790M resistance mutation, has been approved for first-line treatment in four countries including the US and Europe [
40]. To stratify patients in the future, allele frequencies of
EGFR p.T790M subclones in primary tumour samples should be evaluated via ultra-deep parallel sequencing [
41‐
43]. Determination of a clinically relevant cut-off allele frequency could help to distinguish early and late progressors in advance. In future studies, a correlation of
EGFR p.T790M allele frequency with therapeutic results of first-line afatinib treated patients should be performed.
Furthermore, the determination of tumor mutation burden with type and prevalence of resistance mutation acquisition could be of high interest in the future. Since TMB and efficacy of EGFR-Tyrosine kinase inhibitors in patients with EGFR-mutant lung cancers have been investigated and found to be negatively associated in lung cancer patient treated with EGFR TKI [
44].
The present study is the first retrospective analysis based on a patient number as high as 123 first-line patients including 55 afatinib patients. Moreover, it considers the acquisition of resistance mutation under therapy by including only patients with at least 6 months response to EGFR TKI therapy. Furthermore, exclusively first-line treated tumours samples were considered for data evaluation. Therefore, this study avoids falsification of results by e.g. pre-existent resistance mutations per se or inclusions of patients with prior therapies.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.