HSP90 inhibition overcomes EGFR amplification‐induced resistance to third‐generation EGFR‐TKIs

Abstract Background Patients with non‐small cell lung cancer (NSCLC) harboring activating EGFR mutations are sensitive to epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKIs) but inevitably develop resistance to the inhibitors mostly through acquisition of the secondary T790M mutation. Although third‐generation EGFR‐TKIs overcome this resistance by selectively inhibiting EGFR with EGFR‐TKI‐sensitizing and T790M mutations, acquired resistance to third‐generation EGFR‐TKIs invariably develops. Methods Next‐generation sequencing (NGS) and fluorescence in situ hybridization (FISH) analysis were performed in an EGFR T790M‐mutated NSCLC patient who had progressed after a third‐generation EGFR‐TKI, TAS‐121. EGFR‐mutated cell lines were subjected to a cell proliferation assay and western blotting analysis with EGFR‐TKIs and a heat shock protein 90 (HSP90) inhibitor. Results NGS and FISH analysis revealed EGFR amplification in the resistant cancer cells. While EGFR L858R/T90M‐mutated cell line was sensitive to osimertinib or TAS‐121 in vitro, EGFR‐overexpressing cell lines displayed resistance to these EGFR‐TKIs. Western blot analysis showed that EGFR phosphorylation and overexpression of EGFR in cell lines was not suppressed by third‐generation EGFR‐TKIs. In contrast, an HSP90 inhibitor reduced total and phosphorylated EGFR and inhibited the proliferation of resistant cell lines. Conclusions EGFR amplification confers resistance to third‐generation EGFR‐TKIs which can be overcome by HSP90 inhibition. The results provide a preclinical rationale for the use of HSP90 inhibitors to overcome EGFR amplification‐mediated resistance.


INTRODUCTION
Epidermal growth factor receptor (EGFR) mutations are commonly found in non-small cell lung cancer (NSCLC), with a prevalence of 10%-20% in Caucasian patients and 30%-40% in Asian patients with advanced NSCLC. 1,2 Upon mutation of the tyrosine kinase domains of EGFR, EGFR undergoes conformational changes, and its equilibrium is shifted towards a ligand-independent activated state, 3 which results in cell proliferation or survival. 4 Activating EGFR mutations (in-frame deletions in exon 19 and a point mutation in exon 21) trigger tumorigenesis and are a major determinant of susceptibility to EGFR tyrosine kinase inhibitors (EGFR-TKIs). 5 Numerous clinical trials have shown the superior efficacy of first-generation EGFR-TKIs (gefitinib and erlotinib) or second-generation EGFR-TKIs (afatinib) compared with chemotherapy [6][7][8][9][10][11] and have established these agents as the standard of care for advanced EGFRmutated NSCLC.
Despite their marked response to EGFR-TKIs, EGFRmutated NSCLCs inevitably develop resistance to these inhibitors after approximately 8-13 months of treatment. 5 Among the resistance mechanisms, the EGFR T790M mutation is predominant, occurring in approximately half of EGFR-mutated NSCLC cases. 5,12,13 However, this limitation has been overcome by the introduction of third-generation EGFR-TKIs, such as osimertinib. These compounds covalently bind to the C797 residue within the mutant EGFR kinase domain, irreversibly binding to the ATP-binding site while sparing wild-type EGFR. [14][15][16] These characteristics of third-generation EGFR-TKIs have led to their significant efficacy and decreased toxicity in clinical trials, 17,18 and osimertinib has been approved for the treatment of EGFRpositive NSCLC patients. 19 Similar to patients treated with earlier-generation EGFR-TKIs, those receiving third-generation EGFR-TKIs invariably develop drug resistance. Identification of the resistance mechanisms is crucial for improving outcomes in patients with EGFR-mutated NSCLC. Heterogeneous mechanisms underlying resistance to third-generation EGFR-TKIs have been reported; these include the tertiary EGFR C797S mutation; amplification of MET or HER2; mutations in PIK3CA, ALK, or BRAF; and RET fusions. 20 However, the resistance mechanisms are not fully understood and are unknown in 30-50% of cases, 21 necessitating their further investigation.
Heat shock proteins (HSPs) assist in the folding of nascent polypeptides into a functional conformation, thus facilitating protein stability and turnover, which are necessary for the intracellular localization and function of proteins. 22 The HSP90 chaperone machinery, a key regulator of proteostasis, impairs apoptotic signaling in cancer cells. 23 Since EGFR is a client protein for the HSP90 chaperone, a strategy of targeting HSP90 has been evaluated in EGFRmutated NSCLC. 24 While clinical trials have shown the activity of HSP90 inhibitors in NSCLC patients harboring EGFR mutations, 25,26 whether inhibition of HSP90 can overcome acquired resistance to third-generation EGFR-TKIs remains to be determined.
Here, we report a resistance mechanism of EGFR amplification in a patient with EGFR T790M-mutated NSCLC who developed acquired resistance to a third-generation EGFR-TKI, TAS-121. 16 We then investigated the role of EGFR amplification in this resistance in vitro. Furthermore, we evaluated the sensitivity of this tumor to an HSP90 inhibitor (TAS-116) 27 to evaluate the therapeutic possibility of a potent HSP90 inhibitor.

Patient and samples
Tumor samples were obtained by biopsy and autopsy from a patient with metastatic lung adenocarcinoma harboring an EGFR L858R mutation and were analyzed by nextgeneration sequencing (NGS) and/or fluorescence in situ hybridization (FISH).

NGS of clinical samples
NGS (NCC OncoPanel v3, Agilent Technologies) was performed using formalin-fixed, paraffin-embedded (FFPE) samples obtained from a progressive liver lesion during TAS-121 therapy as previously described. 28,29 Sequenced genes are summarized in Table S1.

FISH analysis
We conducted FISH analysis for EGFR with a Vysis EGFR Dual Color Probe-Hyb Set (Abbott Laboratories) using FFPE samples from pre-TAS-121-treatment liver lesions, post-TAS-121-treatment liver lesions, and autopsied lung and liver lesions. EGFR amplification was indicated if the EGFR/CEP signal ratio was >2.0. 30

Cell lines and reagents
The HCC827 and H1975 cell lines (human NSCLC cell lines) were obtained from the American Type Culture Collection (ATCC), and the PC-9 cell line (a human NSCLC cell line) was obtained from the European Collection of Authenticated Cell Cultures (ECACC). The PC9-COR cell line was established from the PC-9 cell line as previously reported. 31 All cell lines were authenticated using the short tandem repeat method and were maintained in RPMI medium (FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% fetal bovine serum (FBS; Cytiva, Marlborough, MA). The EGFR status of the cell lines is summarized in Table S2. All cell lines were used after they were confirmed to be negative for Mycoplasma contamination with a PCR Mycoplasma Detection Kit (TaKaRa Bio) according to the manufacturer's instructions. Erlotinib and osimertinib were obtained from Cayman Chemical Company. TAS-121 and TAS-116 were kindly provided by Taiho Pharmaceutical Co., Ltd.

Cell proliferation assay
Cells were plated in 96-well plates at a density of 2 × 10 3 cells/ well and incubated for 24 h. Cell proliferation was evaluated with a WST-1 assay (TaKaRa Bio) after 72 h of treatment. The absorption of WST-1 was measured at a wavelength of 450 nm with a reference wavelength of 690 nm in a microplate reader. Cell viability was calculated as the ratio of the absorbance value of the treated cells to that of the control cells and expressed as a percentage. Experiments were performed independently in triplicate.

Western blotting
Subconfluent cells were washed with PBS and harvested with M-PER (Thermo Fisher). Whole cell lysates were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. After blocking, the membrane was probed with a primary antibody. After two rinses with TBS-T buffer, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody and washed. Immunoreactions were visualized using an ECL detection system and a LAS-4000 (GE Healthcare). Antibodies are summarized in Table S3. Experiments were performed independently in at least triplicate.

Apoptosis
Apoptosis was assessed using flowcytometry with FITC-annexin V and 7-AAD (Thermo Fisher Scientific). The staining reagents were diluted in accordance with the manufacturer's instructions. The stained cells were analyzed with the LSR Fortessa (BD Biosciences) and FlowJo software (BD Biosciences).

Xenograft studies
Female BALB/c nu/nu nude mice (6-8 weeks old) were purchased from CLEA Japan. H1975 cells, H1975-LR/ TM, and PC9-COR cells (1 × 10 6 ) in 100 μl of RPMI with 100 μl of Matrigel were injected subcutaneously into the backs of mice, and the tumor volume was assessed twice a week using the formula "length × width 2 × 0.5." The mice were grouped when the tumor F I G U R E 1 Clinical course of a 68-year-old woman with metastatic lung adenocarcinoma (cT4N2M1b) harboring an EGFR L858R mutation who received gefitinib and achieved a partial response (PR). Eleven months later, she experienced progressive disease (PD) and received chemotherapy. Liver biopsy of progressing liver metastases indicated a secondary mutation of EGFR T790M in addition to the primary mutation. She was treated with TAS-121 in a phase I trial. Despite the initial response, PD was confirmed by CT which showed regrowth of liver lesions. She died four months after initiation of TAS-121 treatment volume reached approximately 200-500 mm. 3 TAS-121 (12.5 mg/kg/day) and the vehicle were orally administered daily, and TAS-116 (14 mg/kg/day) was orally administered five times a week thereafter. All the mice were maintained under specific pathogen-free conditions in the animal facility of the Institute of Biophysics. The mouse experiments were approved by the Animal Committee for Animal Experimentation of the National F I G U R E 2 EGFR gene status. (a) Next-generation sequencing (NGS) of the EGFR gene. The post-TAS-121-treatment liver sample was analyzed using NGS. The EGFR mutation (left) and copy number gain (right, red) are shown. Blue, wild-type allele; red, T790M-mutated allele. (b) Fluorescence in situ hybridization (FISH). We conducted FISH analyses of EGFR using formalin-fixed, paraffin-embedded (FFPE) samples from the pre-TAS-121-treatment liver lesion, the post-TAS-121-treatment liver lesion, and autopsied lung and liver lesions. Green, CEP; red, EGFR F I G U R E 3 Sensitivities of EGFR-mutated cell lines to EGFR-TKIs. (a) Cell proliferation assay of several EGFR-mutated cell lines under EGFR-TKIs. HCC827 (left), PC-9 (middle), and H1975 (right) cells were plated in 96-well plates at a density of 2 × 10 3 cells/well, and cell proliferation was evaluated with the WST-1 assay following 72 h of treatment. Experiments were independently performed in triplicate, and the means are shown. Black, erlotinib; red, osimertinib; blue, TAS-121. (b) and (c) Western blotting in PC-9 (b) and H1975 cell lines (c). Cells were cultured with the indicated concentrations of EGFR-TKIs for 3 h, and the cell lysates were then subjected to western blot analysis. GAPDH was used as the internal control. Representative data from three independent experiments are shown.

Statistical analysis
Continuous variables were analyzed using a t-test. The relationship between the tumor volume curves were compared using a two-way ANOVA. Statistical analyses were twotailed and performed with the Prism version 7 software (GraphPad Software, Inc., La Jolla, CA, USA). A p-value of less than 0.05 was considered statistically significant.

Clinical course
A 68-year-old woman with metastatic lung adenocarcinoma (cT4N2M1b, stage IV) harboring the EGFR L858R mutation received gefitinib treatment and achieved a partial response (PR) (Figure 1). She experienced progressive disease (PD) 11 months later and subsequently received four cycles of chemotherapy with cisplatin and pemetrexed followed by pemetrexed maintenance therapy. Computed tomography (CT) after six months of chemotherapy showed disease progression with liver metastases. Tumor progression was also noted after completion of one cycle of docetaxel, and rebiopsy of a progressing liver lesion was performed. Tumor genotyping with the peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp method revealed a secondary mutation of EGFR T790M in addition to the primary EGFR L858R mutation. She was enrolled in a phase I trial of TAS-121 (12 mg/day). 32 Despite initial PR, PD was confirmed by CT after three months, indicating progression of liver metastases whereas lung lesions were stable. Rebiopsy of the progressive liver lesion was subsequently performed. She discontinued TAS-121 and received best supportive care. She died four months after initiation of TAS-121 treatment, and an autopsy was performed. NGS of the progressive liver lesion post TAS-121 treatment revealed EGFR amplification (log2 ratio = 2.2). Whereas both the wild-type and T790M-mutated EGFR alleles were amplified, the mutated allele was dominant (Figure 2(a)). In contrast, we were unable to find any typical genetic alterations that can cause resistance to third-generation EGFR-TKIs (i.e., MET amplification or EGFR C797S mutation; Tables S1 and S4). According to the definition of EGFR amplification, which is an EGFR/CEP signal ratio > 2.0 by FISH analysis, 30 no EGFR amplification was identified in the pre-TAS-121-treatment liver lesion (EGFR/CEP signal ratio, 1.7) but was detected in both the rebiopsied post-TAS-121-treatment and autopsied liver lesions (2.1 and 3.0, respectively; Figure 2(b)). EGFR amplification was not observed in the autopsied lung lesions (1.0), which did not progress during TAS-121 treatment. These findings suggest that the patient acquired resistance to TAS-121 owing to the EGFR amplification mainly of the mutated allele.

Third-generation EGFR-TKIs, including TAS-121, effectively inhibit the proliferation of EGFR-mutated NSCLC cells
To verify the efficacy of EGFR-TKIs in EGFR-mutated cell lines, we evaluated sensitivity to the inhibitors in vitro. As expected, the HCC827 and PC-9 cell lines carrying the activating EGFR exon 19 deletion mutation were sensitive to erlotinib (a first-generation EGFR-TKI) and to osimertinib and TAS-121 (third-generation EGFR-TKIs) (Figure 3(a)). The proliferation of H1975 cells harboring the T790M resistance and L858R sensitizing mutations was inhibited by osimertinib and TAS-121 but not by erlotinib (Figure 3(a)). Consistent with this result, western blot analysis showed that osimertinib and TAS-121 reduced phosphorylation of EGFR and the downstream AKT and ERK in the H1975 cell line in a dose-dependent manner as well as in the PC-9 cell line, whereas erlotinib did not decrease phosphorylation in the H1975 cell line (Figure 3(b) and (c)). Apoptosis was significantly induced by all EGFR-TKIs in the PC-9 cell line (Figure 3(d)). In contrast, erlotinib did not induce apoptosis, whereas TAS-121 as well as osimertinib significantly induced apoptosis in the H1975 cell line (Figure 3(e)). These findings confirm that TAS-121, a third-generation EGFR-TKI, overcomes the EGFR T790M-driven resistance to erlotinib by a mechanism similar to that of osimertinib. 16

EGFR amplification meditates resistance to third-generation EGFR-TKIs
The results of NGS data analysis and FISH analysis in our patient suggested that amplification of EGFR, especially the T790M mutant, occurred upon resistance to TAS-121. Thus,  Figure 4(a) shows that H1975-LR/TM cells contained higher levels of phosphorylated and total EGFR than control cells. In the cell proliferation assay, the H1975-LR/ TM cell line was resistant to both TAS-121 and osimertinib (Figure 4(b)). These results were supported by those of western blot analysis; that is, TAS-121 and osimertinib did not inhibit phosphorylation of EGFR and the downstream AKT and ERK in the H1975-LR/TM cell line (Figure 4(c)). Consistently, apoptosis was not induced by TAS-121 and osimertinib in this cell line (Figure 4(d)). These findings suggest that amplification of the mutated EGFR gene mediated the resistance to TAS-121 in this patient.
The image generated by Interactive Genome Viewer showing the possible coemergence of the wild-type EGFR allele prompted us to evaluate the effects of EGFR-TKIs on the PC9-COR cell line with amplification of wild-type EGFR, which was previously established using rociletinib as a clone resistant to erlotinib and third-generation EGFR-TKIs 31 (Figure 2(a)). We confirmed the expression of not mutated but wild-type EGFR in PC9-COR cells ( Figure 5(a)). Accordingly, the PC9-COR cell line displayed resistance to TAS-121 as well as to erlotinib and osimertinib ( Figure 5(b)), and western blot analysis showed that no EGFR-TKI reduced phosphorylation of EGFR and the downstream AKT and ERK ( Figure 5(c)). Apoptosis was not induced by these EGFR-TKIs in this cell line ( Figure 5(d)). These findings are consistent with those of a previous study showing that amplification of wild-type EGFR also mediates resistance to third-generation EGFR-TKIs. 31 Taken together, these results indicate that amplification of both mutated and wild-type EGFR can mediate resistance to third-generation EGFR-TKIs.

HSP90 inhibitor, TAS-116, overcomes EGFR amplification-mediated acquired resistance to third-generation EGFR-TKIs
To explore the possibility that HSP90 inhibition can overcome EGFR amplification-mediated resistance, we analyzed the sensitivities of PC-9, H1975, H1975-LR/TM and PC9-COR cell lines to the HSP90 inhibitor TAS-116 in vitro. Parent cell lines (PC-9 and H1975) were sensitive to TAS-116 ( Figure S1). Additionally, the viability of both H1975-LR/TM and PC9-COR cells was compromised by treatment with the HSP90 inhibitor (Figure 4(b) and Figure 5(b)). Importantly, TAS-116 was highly active against these cell lines at therapeutic concentrations of 1-3 μM. 33,34 In contrast, the combination efficacy of TAS-121 and TAS-116 was not observed in both the H1975 and H1975-LR/TM cell lines ( Figure S1). Consistent with this finding, TAS-116 greatly reduced the levels of both total and phosphorylated EGFR and the downstream AKT and ERK in these cell lines in a concentration-dependent manner (Figure 4(c) and Figure 5(c)). Apoptosis was also significantly induced by TAS-116 (Figures 4(d) and 5(d)). Furthermore, the in vivo effects of TAS-116 against both H1975-LR/TM and PC9-COR tumors that were resistant to TAS-121 were also observed ( Figure 6(a)-(c)). These results provide a preclinical rationale for the use of HSP90 inhibitors to overcome the acquired resistance to third-generation EGFR-TKIs induced by EGFR amplification.

DISCUSSION
In this study, we identified mutated EGFR amplification as a resistance mechanism in a patient with EGFR T790Mpositive NSCLC who experienced PD on a third-generation EGFR-TKI, TAS-121. In vitro, amplification of not only wild-type but also mutated EGFR induced resistance. This is the first case of mutated EGFR amplification-mediated resistance in a patient treated with TAS-121, although this mechanism has been reported in patients treated with other third-generation EGFR-TKIs. 35,36 In addition, TAS-116 overcame this resistance in vitro and in vivo, indicating that HSP90 inhibition is a therapeutic strategy for patients with EGFR amplification-mediated resistance to third-generation EGFR-TKIs.
Acquired resistance has been a challenge in EGFR-TKI therapy. Initial characterization of resistance to earliergeneration EGFR-TKIs identified EGFR T790M mutation as a predominant resistance mechanism and led to the development of third-generation EGFR-TKIs. 5 However, the efficacy of third-generation EGFR-TKIs has been limited by the occurrence of secondary resistance. 20 Numerous studies have shown the heterogeneity of mechanisms underlying resistance to third-generation EGFR-TKIs. In addition to the tertiary resistance EGFR C797S mutation, somatic copy number alterations account for a substantial portion of resistance. 21,37 From our NGS data, we identified EGFR amplification as a resistance mechanism, but no other alterations, including MET amplification and EGFR C797S mutation, were identified as resistance mechanisms. Consistent with our findings, a prior study reported that amplification of mutated EGFR emerged in 39% of plasma samples from patients with acquired resistance to earlier-generation EGFR-TKIs and 9% of those from patients with resistance to a third-generation EGFR-TKI. 38 Amplification of the T790M allele was also confirmed in 23% of biopsy samples from patients with T790M-positive NSCLC at development of resistance to a third-generation EGFR-TKI. 35 Understandably, amplification of wild-type EGFR can confer resistance to third-generation EGFR-TKIs because thirdgeneration EGFR-TKIs selectively inhibit mutated EGFR and allow wild-type EGFR to escape inhibition. 5 Indeed, amplification of wild-type EGFR was reported to decrease the sensitivity of EGFR-mutated cancer cells to thirdgeneration EGFR-TKIs in vitro 31,38,39 and to cause resistance to osimertinib in a patient with T790M-positive NSCLC. 40 Conversely, in the clinical setting, accumulating evidence, including our case, indicates that amplification of mutated EGFR drives resistance to these inhibitors despite the potent inhibition of mutant EGFR by third-generation EGFR-TKIs. 35,38,41 These conflicting results highlight the need for an improved understanding of EGFR amplificationmediated resistance. Here, we found that the overexpression of mutated EGFR outpaced the inhibitory activity of thirdgeneration EGFR-TKIs, providing additional insight into the interplay between EGFR amplification and the response to EGFR-TKIs. In addition, the increasing use of circulating tumor DNA in blood to clarify resistance mechanisms may favor the reporting of amplification of mutated EGFR, because the inevitable contamination by nonmalignant cells hinders clinicians from accurately analyzing the copy numbers of wild-type genes. 31 Thus, the association between the copy numbers of mutated EGFR and EGFR inhibition needs further investigation to determine minimum amplification threshold for induction of resistance, and analyzing not only EGFR mutations but also EGFR copy numbers can facilitate access to optimal therapies in clinical settings.
In our patient, CT on the failure of TAS-121 therapy showed mixed response to the agent; among the multiple lesions in the lung and liver, only liver metastasis progressed, and the lung lesions did not change in size. In the FISH analysis, EGFR amplification was found in the resistant liver lesions but not in the lung lesions, which suggests that heterogeneous cancer cells emerged with resistant EGFR-amplified cancer cells in the liver metastases. Indeed, the results of an ongoing clinical trial raised the possibility that a focal copy number gain occurred subclonally upon the development of osimertinib resistance and was spatially and temporally separated from common resistance mechanisms, such as C797S mutation. 42 Thus, the EGFR amplification in resistant liver lesions in our patient could reflect the evolutionary process of subclones selected by the potent EGFR-inhibitory effects of TAS-121 and demanded to selectively target EGFR amplification to overcome the resistance.
HSP90 inhibitors have exhibited potent antitumor activities in various preclinical models by destabilizing HSP90 client proteins. 43 Importantly, mutated EGFR proteins are particularly reliant on the chaperone activity of HSP90 for their conformational stability and function, 44 which led the H1975-LR/TM cell line harboring the mutated EGFR amplification to have higher sensitivity than the PC9-COR cell line with wild-type EGFR amplification to TAS-116 in our present study. In clinical trials, luminespib, a member of another class of HSP90 inhibitors, exhibited activity against EGFR-mutated NSCLC. 25,26 Consistent with this finding, our present study shows that TAS-116 exhibited efficacies against parent EGFR-mutated cell lines sensitive to EGFR-TKIs. In addition, HSP90 inhibition alone reportedly overcame the MET amplification-or HGF-induced resistance to EGFR-TKIs in vitro. 45,46 Luminespib combined with osimertinib exhibited a marked efficacy for intrinsic resistance to osimertinib by decreasing the phosphorylation of EGFR and MET and downregulating their downstream pathways. 47 Our present study also showed that AKT and ERK, the downstream signals in the EGFR pathway, were degraded by HSP90 inhibition using TAS-116, which supports the use of HSP90 inhibitors to overcome the resistance to EGFR-TKIs.
Our patient received third-generation EGFR-TKI after acquired resistance to gefitinib because at that time no firstline osimertinib therapy had been established. Consequently, we identified EGFR amplification as a resistance mechanism to fourth-line third-generation EGFR-TKI. However, first-line osimertinib therapy is a standard of care in the current clinical setting, and whether EGFR amplification can confer resistance to third-generation EGFR-TKIs regardless of the treatment lines with EGFR-TKIs remains uncertain. In a previous study, EGFR amplification induced resistance to firstline third-generation EGFR-TKIs. 21 An osimertinib-resistant PC9-COR cell line represented the resistance to first-line third-generation EGFR-TKIs, 31 which was overcome by TAS-116 in our study. Thus, HSP90 inhibitors can also overcome EGFR amplification-mediated resistance to first-line thirdgeneration EGFR-TKIs.
HSP90 inhibitors have shown limited efficacy as single agents. 43 Undesirable off-target and/or HSP90-related adverse events could account for this discrepancy via the need to limit the drug concentrations to levels insufficient to efficiently suppress intratumoral HSP90 activity. 24 The most common adverse event in patients receiving HSP90 inhibitors was visual disorders due to sustained HSP90 inhibition in the retina. 48 TAS-116 possesses a distinct advantage over other HSP90 inhibitors, as its distribution in retinal tissue is lower than that in plasma, and it is rapidly eliminated from the retina. 27 Indeed, eye disorders were not clinically significant in trials for patients with advanced solid tumors. 33,34 Therefore, further studies should focus on TAS-116 as a promising therapeutic option for EGFR-mutated lung cancer. In particular, patient-derived experiments (xenograft model and patient-derived cell line or organoid) would be an alternative method to validate their efficacy.
Another strategy to overcome EGFR amplificationmeditated resistance is the addition of cetuximab, a human-mouse chimeric antibody that binds to the extracellular domain of EGFR, to third-generation EGFR-TKIs. The efficacy of cetuximab combined with afatinib, a second-generation EGFR-TKI, has been reported in previous studies. [49][50][51] By inhibiting EGF-induced activation of wild-type EGFR in PC9-COR cell line 31 or dimerization of mutated EGFR induced by EGFR-TKI in erlotinibresistant EGFR-mutated cell lines, 52 cetuximab can enhance the inhibition of third-generation EGFR-TKIs. However, the addition of cetuximab to afatinib did not improve the outcomes in previously untreated EGFRmutant NSCLC patients but led to greater toxicity, including a 72% incidence rate of grade ≥ 3 treatment-related adverse events. 53 In summary, this study demonstrated that mutated EGFR amplification led to resistance to a third-generation EGFR-TKI, TAS-121, in a patient with EGFR T790Mpositive NSCLC. Targeting the HSP90 chaperone using TAS-116 overcame this resistance in vitro and in vivo, indicating a preclinical rationale for the use of HSP90 inhibitors in patients with EGFR amplification-mediated resistance to third-generation EGFR-TKIs. Additional investigations into EGFR amplification-mediated resistance and the efficacy and safety of HSP90 inhibition are warranted for the development of optimal therapies.