Afatinib Resistance Research Articles

Clinical efficacy and identification of factors confer resistance to afatinib (tyrosine kinase inhibitor) in EGFR-overexpressing esophageal squamous cell carcinoma

Epidermal growth factor receptor (EGFR) is reportedly overexpressed in most esophageal squamous cell carcinoma (ESCC) patients, but anti-EGFR treatments offer limited survival benefits. Our preclinical data showed the promising antitumor activity of afatinib in EGFR-overexpressing ESCC. This proof-of-concept, phase II trial assessed the efficacy and safety of afatinib in pretreated metastatic ESCC patients (n = 41) with EGFR overexpression (NCT03940976). The study met its primary endpoint, with a confirmed objective response rate (ORR) of 39% in 38 efficacy-evaluable patients and a median overall survival of 7.8 months, with a manageable toxicity profile. Transcriptome analysis of pretreatment tumors revealed that neurotrophic receptor tyrosine kinase 2 (NTRK2) was negatively associated with afatinib sensitivity and might serve as a predictive biomarker, irrespective of EGFR expression. Notably, knocking down or inhibiting NTRK2 sensitized ESCC cells to afatinib treatment. Our study provides novel findings on the molecular factors underlying afatinib resistance and indicates that afatinib has the potential to become an important treatment for metastatic ESCC patients.

Clinical phase II trial shows that combining osimertinib and afatinib resistance EGFR recurrent mutation in EGFR-mutant lung cancer

Treatment options for patients with non-small-cell lung cancer (NSCLC) with EGFR mutations are limited due to resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). Osimertinib or afatinib alone, in a preclinical model, created drug-resistant clones with EGFR secondary mutations, but their combination inhibited the emergence of these mutations. In a Phase II trial, we looked into the alternating-dose therapy of osimertinib and afatinib in patients with EGFR-mutant NSCLC. Patients with stage IV NSCLC with an activating EGFR mutation who had never received treatment were included. Every eight weeks, osimertinib (80 mg/day) and afatinib (20 mg/day) were given in alternate cycles. Utilising circulating tumour DNA collected both before and after therapy, genomic analysis was carried out. The median progression-free survival among the 50 enrolled patients was 21.3 months. A total of 70.3% of respondents responded. Overall median survival was not attained. 35 plasma samples were acquired after the development of resistance; 5 of these samples displayed an elevated MET gene copy number and 3 displayed a BRAF mutation. However, no secondary EGFR mutation was found. The effectiveness of our approach was comparable to that of osimertinib alone, as had been observed in untreated advanced NSCLC patients with EGFR mutations in the past. The treatment may stop the emergence of EGFR secondary mutations that lead to medication resistance, despite the small sample size. To determine the importance of this treatment, more research is required.

Identification and Validation of Afatinib Potential Drug Resistance Gene BIRC5 in Non-Small Cell Lung Cancer.

IntroductionResistance to second-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), afatinib, is the most significant challenge in the clinical management of non-small cell lung cancer (NSCLC), and the underlying mechanisms remain unclear.MethodsGenomic signatures that may confer afatinib resistance in NSCLC were identified via data mining of public databases and integrative bioinformatic analyses. Furthermore, acquired afatinib-resistant lung adenocarcinoma cell lines (HCC827 AR) were established by long-term exposure under afatinib in vitro for stepwise escalation. The expression of baculovirus IAP repeat protein 5 (BIRC5) was detected by western blot, and cellular viability of HCC827 AR was determined by CCK8.ResultsThrough integrative bioinformatic analyses of public datasets, overexpression of baculovirus IAP repeat protein 5 (BIRC5) was identified in both afatinib-resistant NSCLC cells and tissues, and BIRC5 overexpression was positively correlated with lymph node metastasis as well as pathological stage in NSCLC. Furthermore, NSCLC patients with BIRC5 overexpression showed poor survival outcomes. Immune infiltration analysis suggested that BIRC5 expression was significantly inversely correlated with tumor-infiltrating cell numbers and immune biomarker expression in NSCLC. The functions of genes co-expressed with BIRC5 were mainly enriched in cell cycle mitotic phase transition, double-strand break repair, and negative regulation of the cell cycle process signaling pathway. In addition, overexpression of BIRC5 protein was detected in afatinib-resistant cells by western blot, while BIRC5-expressing cells treated with BIRC5 inhibitor, YM155, were sensitive to afatinib.ConclusionsIn this study, we showed that overexpression of BIRC5 resulted in resistance to afatinib in NSCLC and BIRC5-specific inhibitors may overcome the resistant phenotype, indicating that dysregulation of the apoptotic cell death pathway may be the key mechanism underlying TKI resistance in the development of NSCLC.

Afatinib treatment response in advanced lung adenocarcinomas harboring uncommon mutations

BackgroundEpidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have improved the prognosis of mutant lung cancer; however, the clinical application value of TKIs for nonclassical EGFR mutation is unclear, especially for patients with rare uncommon mutations.MethodsA retrospective study based on electronic medical records was conducted to collect data on the effectiveness of afatinib in patients with stage IIIB/IV lung adenocarcinoma (LUAD) bearing uncommon mutations between January 2017 and January 2021.ResultsForty‐two patients with uncommon mutations treated with afatinib were enrolled. The objective response rate (ORR) was 50.0% (10 of 20 patients). The median time to treatment failure (TTF) was 11.7 months (95% confidence interval = 8.5–18.3 months). Of the 42 patients, the median TTF was 15.0, 11.7, and 16.6 months in patients with Gly719Xaa (G719X), Ser768Ile (S768I), and Leu861Gln (L861Q) mutations, respectively. In patients with the rare uncommon mutation, the median TTF was 10.0 months, and the ORR was 50.0%. Afatinib demonstrated clinical activity across a set type of specific rare uncommon mutations, including EGFR L747P, A767_V769dup, and L833V/H835L, with a case having a TTF of more than 1 year. Molecular profiling reports of 16 afatinib‐resistant biopsy samples were available, and the secondary T790M mutation was detected in one patient with L833V/H835L mutation and one harboring S768I/L858R mutation.ConclusionsOur findings suggested that afatinib is effective in patients with uncommon mutations. Mechanisms of afatinib resistance vary and need further investigation.

Afatinib as a Potential Therapeutic Option for Patients With NSCLC With EGFR G724S

IntroductionEGFR G724S has been described to mediate resistance to first- and third-generation EGFR tyrosine kinase inhibitors (TKIs). In vitro experiments have provided compelling evidence that G724S retains sensitivity for afatinib. Nevertheless, limited data have reported the clinical efficacy of afatinib in patients with NSCLC harboring G724S mutation. MethodsWe identified 52 patients with NSCLC with EGFR G724S from an inhouse database and comprehensively profiled their concurrent mutation statuses. Treatments and clinical outcomes were also collected. ResultsOf 52 G724S-positive patients, 39 harbored concomitant EGFR exon 19 deletion (19del), and all 37 of the 39 patients who had available clinical data were detected with a G724S mutation after receiving EGFR TKIs. A rare variant of 19del E746_S752delinsV co-occurred with G724S the most frequently (n = 29), whereas 7 of 10 patients with concomitant EGFR exon 20 mutation were TKI treatment naive. S768I was the most common mutation in exon 20 (n = 7). One patient harbored a concomitant EGFR exon 21 mutation, and two lacked co-occurring EGFR mutations. A total of 23 patients provided valid clinical outcome data, of whom eight were treated with afatinib after the emergence of G724S, whereas 15 received non-afatinib treatment (alternative EGFR TKI, chemotherapy, or best supportive care). The disease control rate in afatinib-treated patients (n = 8) reached 100% with a median progression-free survival of 4.5 months, significantly longer than that of non–afatinib-treated (n = 15, 1.7 mo, hazard ratio [HR] = 0.32, p = 0.037) and alternative EGFR TKI-treated (n = 11, 1.8 mo, HR = 0.28, p = 0.042) patients. In the subset who had progressed on osimertinib, afatinib also yielded a superior progression-free survival (6.2 mo) than non-afatinib therapies (1.0 mo, HR = 0.04, p = 0.005) and alternative EGFR TKIs (1.8 mo, HR = 0.06, p = 0.033). Analysis of acquired mutations at afatinib progression revealed re-emergence of EGFR T790M or MET amplification as the potential mechanism of afatinib resistance. ConclusionsEGFR G724S emerges as a resistant mutation against EGFR TKI preferentially in the context of a rare variant of 19del, whereas it might mediate differential mechanisms in the context of exon 20 mutation. We also found that afatinib could be a potential therapeutic option for patients with NSCLC with G724S.

CALM1 promotes progression and dampens chemosensitivity to EGFR inhibitor in esophageal squamous cell carcinoma

BackgroundCalmodulin1 (CALM1) has been identified as one of the overexpression genes in a variety of cancers and EGFR inhibitor have been widely used in clinical treatment but it is unknown whether CALM1 and epidermal growth factor receptor (EGFR) have a synergistic effect in esophageal squamous cell carcinoma (ESCC). The aim of the present study was to explore the synergistic effects of knock-out CALM1 combined with EGFR inhibitor (Afatinib) and to elucidate the role of CALM1 in sensitizing the resistance to Afatinib in ESCC.MethodImmunohistochemistry (IHC) and qRT-PCR were used to examine the expression of CALM1 and EGFR in ESCC tissues. Kaplan–Meier survival analysis was used to analyze the clinical and prognostic significance of CALM1 and EGFR expression in ESCC. Furthermore, to evaluate the biological function of CALM1 in ESCC, the latest gene editing technique CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats)was applied to knockout CALM1 in ESCC cell lines KYSE150, Eca109 and TE-1. MTT, flow cytometry, Transwell migration, scratch wound-healing and colony formation assays were performed to assay the combined effect of knock-out CALM1 and EGFR inhibitor on ESCC cell proliferation and migration. In addition, nude mice xenograft model was used to observe the synergistic inhibition of knock-out CALM1 and Afatinib.ResultsBoth CALM1 and EGFR were found to be significantly over-expressed in ESCC compared with paired normal control. Over-expressed CALM1 and EGFR were significantly associated with clinical stage, T classification and poor overall prognosis, respectively. In vitro, the combined effect of knock-out CALM1 mediated by the lentivirus and EGFR inhibitor was shown to be capable of inhibiting the proliferation, inducing cell cycle arrest at G1/S stage and increasing apoptosis of KYSE-150 and Eca109 cells; invasion and migration were also suppressed. In vivo, the results of tumor weight and total fluorescence were markedly reduced compared with the sgCtrl-infected group and sgCAML1 group.ConclusionOur data demonstrated that knock-out of CALM1 could sensitize ESCC cells to EGFR inhibitor, and it may exert oncogenic role via promotion of EMT. Taken together, CALM1 may be a tempting target to overcome Afatinib resistance.

Celastrol acts synergistically with afatinib to suppress non-small cell lung cancer cell proliferation by inducing paraptosis.

Non-small cell lung cancer (NSCLC) with wild-type epidermal growth factor receptor (EGFR) is intrinsic resistance to EGFR-tyrosine kinase inhibitors (TKIs), such as afatinib. Celastrol, a natural compound with antitumor activity, was reported to induce paraptosis in cancer cells. In this study, intrinsic EGFR-TKI-resistant NSCLC cell lines H23 (EGFR wild-type and KRAS mutation) and H292 (EGFR wild-type and overexpression) were used to test whether celastrol could overcome primary afatinib resistance through paraptosis induction. The synergistic effect of celastrol and afatinib on survival inhibition of the NSCLC cells was evaluated by CCK-8 assay and isobologram analysis. The paraptosis and its modulation were assessed by light and electron microscopy, Western blot analysis, and immunofluorescence. Xenografts models were established to investigate the inhibitory effect of celastrol plus afatinib on the growth of the NSCLC tumors in vivo. Results showed that celastrol acted synergistically with afatinib to suppress the survival of H23 and H292 cells by inducing paraptosis characterized by extensive cytoplasmic vacuolation. This process was independent of apoptosis and not associated with autophagy induction. Afatinib plus celastrol-induced cytoplasmic vacuolation was preceded by endoplasmic reticulumstress and unfolded protein response. Accumulation of intracellular reactive oxygen speciesand mitochondrial Ca2+ overload may be initiating factors of celastrol/afatinib-induced paraptosis and subsequent cell death. Furthermore, Celastrol and afatinib synergistically suppressed the growth of H23 cell xenograft tumors in vivo. The data indicate that a combination of afatinib and celastrol may be a promising therapeutic strategy to surmount intrinsic afatinib resistance in NSCLC cells.

Molecular profiling of afatinib-resistant non-small cell lung cancer cells in vivo derived from mice

Non-small-cell lung cancer (NSCLC) is a leading cause of cancer-related death worldwide. NSCLC patients with overexpressed or mutated epidermal growth factor receptor (EGFR) related to disease progression are treated with EGFR-tyrosine kinase inhibitors (EGFR-TKIs). Acquired drug resistance after TKI treatments has been a major focus for development of NSCLC therapies. This study aimed to establish afatinib-resistant cell lines from which afatinib resistance-associated genes are identified and the underlying mechanisms of multiple-TKI resistance in NSCLC can be further investigated. Nude mice bearing subcutaneous NSCLC HCC827 tumors were administered with afatinib at different dose intensities (5–100 mg/kg). We established three HCC827 sublines resistant to afatinib (IC50 > 1 μM) with cross-resistance to gefitinib (IC50 > 5 μM). cDNA microarray revealed several of these sublines shared 27 up- and 13 down-regulated genes. The mRNA expression of selective novel genes — such as transmembrane 4 L six family member 19 (TM4SF19), suppressor of cytokine signaling 2 (SOCS2), and quinolinate phosphoribosyltransferase (QPRT) — are responsive to afatinib treatments only at high concentrations. Furthermore, c-MET amplification and activations of a subset of tyrosine kinase receptors were observed in all three resistant cells. PHA665752, a c-MET inhibitor, remarkably increased the sensitivity of these resistant cells to afatinib (IC50 = 12–123 nM). We established afatinib-resistant lung cancer cell lines and here report genes associated with afatinib resistance in human NSCLC. These cell lines and the identified genes serve as useful investigational tools, prognostic biomarkers of TKI therapies, and promising molecule targets for development of human NSCLC therapeutics.

Abstract 1986: Mutation analysis in cell-free DNA upon development of resistance to afatinib

Abstract Introduction: EGFR-mutant lung cancers are genomically heterogeneous in terms of compound EGFR mutations and mutations in other genes. The purpose of this study was to explore gene mutations in circulating cell-free DNA that are related to resistance to afatinib. Patients and methods: Patients with EGFR-mutant non-small cell lung cancer who received afatinib monotherapy were enrolled in this trial. Plasma samples were longitudinally collected before afatinib treatment, during treatment, and at the time of development of afatinib resistance. Written informed consent was obtained from all patients. Cell-free DNA was extracted from plasma using the QIAamp Circulating Nucleic Acid Kit (QIAGEN). Mutation profiles were determined by next-generation sequencing and analyzed using a human lung cancer panel (QIAGEN). Results: A total of 71 patients were enrolled in this study. Plasma samples both before treatment and at the time of resistance to afatinib were collected from 21 patients. Although currently under analysis, the mutation profiles determined by next-generation sequencing revealed the following changes. 1) The number of gene mutations per patient that newly appeared at resistance development ranged from 0 to 7. 2) The allele frequency of the T790M EGFR mutation, a mutation responsible for resistance to EGFR tyrosine kinase inhibitors, was increased after development of afatinib resistance in approximately half of the patients. Conclusions: The T790M allele frequency tended to increase after the development of resistance to afatinib. Some mutations were detected after resistance to afatinib. The role of this mutation needs to be investigated in a further study. Citation Format: Nanao Terada, Hideharu Kimura, Mako Miyakawa, Hayato Koba, Taro Yoneda, Takayuki Takeda, Hiroki Shirasaki, Kazuhiko Shibata, Koichi Nishi, Koichi Takayama, Kazuo Kasahara. Mutation analysis in cell-free DNA upon development of resistance to afatinib [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1986.

Dissecting the m6A methylation affection on afatinib resistance in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer deaths. Afatinib is the first-line anti-cancer agent for treatment of NSCLC. However, unexpected resistance has been a major obstacle for its clinical efficacy. In this study, we dissected afatinib resistance from the perspective of N6-Methyladenosine (m6A) modification. First, we depicted the m6A modification profiles for the afatinib resistant and sensitive NSCLC cell lines (H1299 and A549). We found that the sum enrichment scores of the resistant cell line (H1299) was much higher than that of the sensitive cell line (A549). Next, we identified the functionally m6A-modified genes, which were the intersection of the differentially m6A methylated genes and the differentially expressed genes between H1299 and A549, as well as negative correlation between m6A modification levels and gene expression levels. In addition, functional enrichment analysis of the functionally m6A-modified genes indicated that m6A methylation might modify cell cycle to affect afatinib response. Furthermore, the functionally m6A-modified genes were over-represented in the putative drug resistance-associated genes and the FDA-approved drug targets, and had significantly higher average degree and clustering coefficient than other genes in protein-protein interaction (PPI) network. We also identified five network modules, which were all related to drug resistance functions. Finally, survival analysis demonstrated that m6A modification could affect prognosis of NSCLC patients. In conclusion, we conducted a first attempt to dissect m6A methylation affection on afatinib resistance in NSCLC, and brought inspiration for the study of epigenetic roles in drug resistance.

MET as resistance factor for afatinib therapy and motility driver in gastric cancer cells.

The therapeutic options for advanced gastric cancer are still limited. Several drugs targeting the epidermal growth factor receptor family have been developed. So far, the HER2 antibody trastuzumab is the only drug targeting the HER-family that is available to gastric cancer patients. The pan-HER inhibitor afatinib is currently investigated in clinical trials and shows promising results in cell culture experiments and patient-derived xenograft (PDX) models. However, some cell lines do not respond to afatinib treatment. The determination of resistance factors in these cell lines can help to find the best treatment option for gastric cancer patients. In this study, we analyzed the role of MET as a resistance factor for afatinib therapy in a gastric cancer cell line. MET expression in afatinib-resistant MET-amplified Hs746T cells was reduced by means of siRNA transfection. The effects of MET knockdown on signal transduction, cell proliferation and motility were examined. In addition to the manual assessment of cell motility, a computational motility analysis involving parameters such as (approximate) average speed, displacement entropy or radial effectiveness was realized. Moreover, the impact of afatinib was compared between MET knockdown cells and control cells. MET knockdown in Hs746T cells resulted in impaired signal transduction and reduced cell proliferation and motility. Moreover, the afatinib resistance of Hs746T cells was reversed after MET knockdown. Therefore, the amplification of MET is confirmed as a resistance factor in gastric cancer cells. Whether MET is a useful resistance marker for afatinib therapy or other HER-targeting drugs in patients should be investigated in clinical trials.

Secreted Phosphoprotein 1 (SPP1) Contributes to Second-Generation EGFR Tyrosine Kinase Inhibitor Resistance in Non-Small Cell Lung Cancer.

Second-generation irreversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), afatinib, has been approved for treating EGFR mutant lung cancer patients, but the mechanism of acquired resistance to afatinib has not been well studied. In this study, we established afatinib acquired resistant cell lines. Gene array technology was used to screen changes in gene expression between afatinib-resistant lung cancer cells and parental cells. Our results showed that secreted phosphoprotein 1 (SPP1) was significantly increased in afatinib-resistant lung cancer cells. To study the effect of SPP1 on afatinib resistance, siSPP1 was used to knock down SSP1 in afatinib-resistant lung cancer cells. Then sensitivity to afatinib and invasive ability were studied. We found that knockdown of SPP1 increased sensitivity of lung cancer cells to afatinib and decrease the ability of invasion. Of clinical significance, we found that SSP1 was upregulated in lung cancer tissues compared with adjacent normal tissues, and low level of SSP1 was strongly associated with better overall survival. Our results suggest that SPP1 enhanced the second-generation EGFR TKI resistance in lung cancer, and inhibiting SPP1 might be a therapeutic target to overcome afatinib resistance.

Abstract 2113: KRAS amplification mediates resistance to osimertinib in acquired afatinib-resistant NSCLC harboring exon 19 deletion/T790M in EGFR

Abstract Background: The second mutation of T790M in epidermal growth factor receptor (EGFR) exon 20 mediates resistance to first- and second-generation EGFR tyrosine kinase inhibitors (TKIs; gefitinib, erlotinib, and afatinib). The critical T790M mutation in EGFR has facilitated the development of third-generation mutation-selective EGFR TKIs (rociletinib and osimertinib). We previously reported heterogeneous afatinib resistance in lung adenocarcinoma PC-9 cells, harboring 15bp deletions in EGFR exon 19. Among the afatinib-resistant PC-9 cells, we noted the emergence of T790M in EGFR exon 20 and responses to third-generation EGFR-TKIs. Here, we used afatinib-resistant lung adenocarcinoma PC-9 cells, called AfaR, harboring the exon 19 deletion/T790M in EGFR. Purpose: To identify novel resistance mechanisms in post-afatinib treatment, two osimertinib-resistant cell lines, OsiR1 and OsiR2, were established using increasing concentrations of osimertinib by stepwise dose escalation up to 1 μmol/L over 10 to 12 months. Results: A linear and reversible KRAS amplification and overexpression with increased osimertinib concentrations in OsiR1 and OsiR2 cells, respectively, was detected. OsiR1 cells maintained osimertinib resistance with KRAS amplification after osimertinib withdrawal for 2 months. After osimertinib withdrawal for 2 months, OsiR2 cells exhibited KRAS attenuation and osimertinib sensitivity was entirely recovered in vitro and in vivo. Phospho-EGFR (Y1068) and growth factor receptor-bound protein 2 (GRB2)/son of sevenless homolog 1 (SOS1) complex was found to mediate osimertinib resistance in OsiR1 cells with sustained KRAS activation. The association of phosphorylated EGFR with the GRB2/SOS1 complex leads to KRAS activation. After 2 months of osimertinib withdrawal, the GRB2/SOS1 complex was dissociated, and the EGFR signal but not the GRB2/SOS1 signal was activated. Concomitant inhibition of mitogen-activated protein kinase kinase and EGFR could overcome osimertinib-resistance in OsiR1 cells. Conclusion: We characterized novel acquired resistance mechanisms for third-generation EGFR-TKI, osimertinib, providing insights into the development of novel treatment strategies. Citation Format: Toshimitsu Yamaoka, Ohba Motoi, Yasunari Kishino, Sojiro Kusumoto, Junji Tsurutani, Tohru Ohmori. KRAS amplification mediates resistance to osimertinib in acquired afatinib-resistant NSCLC harboring exon 19 deletion/T790M in EGFR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2113.

Acquired resistance mechanisms to afatinib in HER2-amplified gastric cancer cells.

Cancer treatment, especially that for breast and lung cancer, has entered a new era and continues to evolve, with the development of genome analysis technology and the advent of molecular targeted drugs including tyrosine kinase inhibitors. Nevertheless, acquired drug resistance to molecular targeted drugs is unavoidable, creating a clinically challenging problem. We recently reported the antitumor effect of a pan‐HER inhibitor, afatinib, against human epidermal growth factor receptor 2 (HER2)‐amplified gastric cancer cells. The purpose of the present study was to identify the mechanisms of acquired afatinib resistance and to investigate the treatment strategies for HER2‐amplified gastric cancer cells. Two afatinib‐resistant gastric cancer cell lines were established from 2 HER2‐amplified cell lines, N87 and SNU216. Subsequently, we investigated the molecular profiles of resistant cells. The activation of the HER2 pathway was downregulated in N87‐derived resistant cells, whereas it was upregulated in SNU216‐derived resistant cells. In the N87‐derived cell line, both MET and AXL were activated, and combination treatment with afatinib and cabozantinib, a multikinase inhibitor that inhibits MET and AXL, suppressed the cell growth of cells with acquired resistance both in vitro and in vivo. In the SNU216‐derived cell line, YES1, which is a member of the Src family, was remarkably activated, and dasatinib, a Src inhibitor, exerted a strong antitumor effect in these cells. In conclusion, we identified MET and AXL activation in addition to YES1 activation as novel mechanisms of afatinib resistance in HER2‐driven gastric cancer. Our results also indicated that treatment strategies targeting individual mechanisms of resistance are key to overcoming such resistance.

Cx32 mediates norepinephrine-promoted EGFR-TKI resistance in a gap junction-independent manner in non-small-cell lung cancer.

The second-generation EGFR-TKI Afatinib is an irreversible ErbB family blocker used to treat patients with non-small-cell lung cancer (NSCLC). Unfortunately, resistance to this drug develops over time, and patients are always under great psychological pressure. A previous study showed that chronic stress hormones participate in EGFR-TKI resistance via β2 -AR signaling via an IL-6 dependent mechanism. Our study further explores a novel potential underlying mechanism. In the present study, we show that the stress hormone norepinephrine (NE) promotes Afatinib resistance by upregulating Cx32 expression. Furthermore, we, for the first time, find that Cx32 is a target gene for transcription factor CREB and NE enhances Cx32 mRNA expression by activation of CREB. We also demonstrate that Cx32 promotes Afatinib resistance by decreasing the degradation of EGFR-TKI resistance-associated proteins (MET, IGF-1R) and by increasing their transcription levels. Together, these results reveal that the stress hormone NE accelerates Afatinib resistance by increasing the expression of Cx32, which augments MET and IGF-1R levels in cancer cells and provides a promising therapeutic strategy against EGFR-TKI Afatinib resistance in NSCLC.

Afatinib in the treatment of advanced NSCLC with EGFR mutation: An observational real-world study.

e20518 Background: Compared with the first generation of EGFR-TKI, Afatinib has a broader spectrum of activity and can better inhibit tumor growth. At present, the therapeutic effects of Afatinib varies on patients with different EGFR mutations and studies on the mechanism of acquired Afatinib resistance are limited. The aim of this study was to evaluate the efficacy and safety of Afatinib in the treatment of EGFR-mutation NSCLC who hadn’t received EGFR-TKI in the past. Methods: 88 patients with advanced NSCLC who had EGFR mutations were enrolled and given oral 40 mg Afatinib daily until disease progression or adverse events that could not be handled by dosage reduction. Efficacy and safety in different subgroups were analyzed. Results: Among 88 patients, the common mutation rate of EGFR was 79.5%, and the rare mutation rate was 20.5%. The objective remission rate (ORR) was 54.5%, and the disease control rate (DCR) was 92.0%. The median progression-free survival (PFS) was 14.2 months (95% CI 11.4-18.5), 27 patients (30.7%) continued Afatinib treatment after tumor progression, and the median time to progression of clinical symptoms (TTSP) was 16.3 months (95% CI 12.7-19.3). A total of 13 patients (14.8%) reduced Afatinib dosage of due to adverse events. Subgroup analysis showed that Afatinib had better effect on patients harboring common EGFR mutation but not exon 20 mutation, and was not affected by brain metastasis, dosage and lines of therapy. Among patients who progressed on Afatinib, 65.4% harbored T790M mutation. Most T790M patients received the third generation EGFR-TKI including AZD9291 and oxetinib treatment. The most common adverse reactions were diarrhea, rash, paronychia and stomatitis. Conclusions: Afatinib showed good efficacy and tolerance in advanced NSCLC patients with EGFR mutations. Continuation of Afatinib treatment after cancer progression could delay the progression of disease symptoms. The third generation EGFR-TKI could become a treatment option after Afatinib resistance. These findings reflected the real-world clinical practice of Afatinib.

Synergistic Growth Inhibition by Afatinib and Trametinib in Preclinical Oral Squamous Cell Carcinoma Models.

Given that aberrant activation of epidermal growth factor receptor family receptors (ErbB) is a common event in oral squamous cell carcinoma, and that high expression of these receptor proteins is often associated with poor prognosis, this rationalizes the approach of targeting ErbB signaling pathways to improve the survival of patients with oral squamous cell carcinoma. However, monotherapy with the ErbB blocker afatinib has shown limited survival benefits. This study was performed to identify mechanisms of afatinib resistance and to explore potential afatinib-based combination treatments with other targeted inhibitors in oral squamous cell carcinoma. We determined the anti-proliferative effects of afatinib on a panel of oral squamous cell carcinoma cell lines using a crystal violet-growth inhibition assay, click-iT 5-ethynyl-2'-deoxyuridine staining, and cell-cycle analysis. Biochemical assays were performed to study the underlying mechanism of drug treatment as a single agent or in combination with the MEK inhibitor trametinib. We further evaluated and compared the anti-tumor effects of single agent and combined treatment by using oral squamous cell carcinoma xenograft models. In this study, we showed that afatinib inhibited oral squamous cell carcinoma cell proliferation via cell-cycle arrest at the G0/G1 phase, and inhibited tumor growth in xenograft mouse models. Interestingly, we demonstrated reactivation of the mitogen-activated protein kinase (ERK1/2) pathway in vitro, which possibly reduced the effects of ErbB inhibition. Concomitant treatment of oral squamous cell carcinoma cells with afatinib and trametinib synergized the anti-tumor effects in oral squamous cell carcinoma-bearing mouse models. Our findings provide insight into the molecular mechanism of resistance to afatinib and support further clinical evaluation into the combination of afatinib and MEK inhibition in the treatment of oral squamous cell carcinoma.

Anti-tumor activity of Shikonin against afatinib resistant non-small cell lung cancer via negative regulation of PI3K/Akt signaling pathway.

Acquired resistance of afatinib is a significant challenge for non-small cell lung cancer (NSCLC) therapy and the mechanisms remain unclear. Aberrant activation of epidermal growth factor receptor (EGFR)-dependent downstream pathways, especially phosphatidylinositol-3-kinases/protein kinase B (PI3K/Akt) signaling pathway has been reported to be involved in the occurrence of afatinib resistance. Developing effective anti-cancer agents to overcome afatinib resistance by targetting PI3K/Akt signaling pathway will be a potential strategy for NSCLC treatment. Shikonin is a naphthoquinone compound isolated from the roots of Lithospermum erythrorhizon. In the present study, the anti-cancer activity of Shikonin was evaluated on afatinib-resistant NSCLC in vitro and in vivo. The data showed that Shikonin inhibited the proliferation and induced apoptosis of afatinib-resistant NSCLC cell line by activating apoptosis signaling pathway and negatively regulating PI3K/Akt signaling pathway. These results revealed that Shikonin was a potential apoptosis inducer in afatinib-resistant NSCLC and a promising candidate for treating patients clinically.

Exploration of resistance mechanisms for epidermal growth factor receptor‐tyrosine kinase inhibitors based on plasma analysis by digital polymerase chain reaction and next‐generation sequencing

Liquid biopsy offers a potential alternative to tissue biopsy for detection of genetic alterations in cancer, and it has been introduced into clinical practice to detect the tyrosine kinase inhibitor (TKI) resistance‐conferring T790M mutation of epidermal growth factor receptor (EGFR) in patients with non‐small‐cell lung cancer (NSCLC). We prospectively collected tumor and plasma samples from 25 NSCLC patients who harbored activating mutations of EGFR and experienced failure of treatment with afatinib. The samples were analyzed by digital PCR (dPCR) and next‐generation sequencing (NGS). T790M was detected in plasma with a respective sensitivity and specificity of 83.3% and 70.0% by dPCR and 50.0% and 70.0% by NGS relative to analysis of corresponding tumor samples. Quantitation of T790M based on the ratio of the number of T790M alleles to that of activating mutation alleles (T/A ratio) improved the specificity of plasma analysis to 100% for both dPCR and NGS without a reduction in sensitivity. Although several afatinib resistance mechanisms other than T790M—including copy number gain of NRAS or MET—were identified in tumor samples, the corresponding genetic alterations were not detected in plasma. TP53 mutations were frequently identified in plasma and tumor samples, with most such mutations also having been detected before afatinib treatment. The presence of de novo TP53 mutations was associated with reduced progression‐free survival. Quantitation of T790M in plasma is thus a clinically relevant approach to determine the T790M status of tumors. In addition, genetic alterations coexisting with EGFR mutations can affect the efficacy of EGFR‐TKI treatment.

P2.13-18 A Multicenter Prospective Biomarker Study to Explore Mechanisms of Afatinib Resistance Based on Digita PCR and Next-Generation Sequencing

Afatinib is an oral irreversible blocker of ErbB-family kinases and shows a pronounced anti-tumor efficacy for advanced non–small cell lung cancer (NSCLC) positive for activating mutations of EGFR. We applied digital polymerase chain reaction (dPCR) and next-generation sequencing (NGS) to explore mechanisms of afatinib resistance. Eligible patients had advanced lung adenocarcinoma with EGFR activating mutations. Tumor and plasma samples were collected before afatinib treatment and after treatment failure with disease progression (systemic progressive disease, SPD). DNA from the samples was analyzed by dPCR and NGS. Thirty-five patients were enrolled, with a median follow-up time of 15.8 months. Among 25 patients with SPD, tumor, plasma, or both samples were available for 18, 23, and 16 individuals, respectively. dPCR and NGS detected EGFR T790M mutation in 13 (56.5%) and 11 (47.8%) of 23 plasma samples at SPD, with sensitivity and specificity compared with tumor samples being 83.3% and 70.0% (dPCR) and 50.0% and 70.0% (NGS), respectively. Applying the ratio of the number of T790M alleles to that of activating mutations (T/A) for determination of the T790M positivity improved the sensitivity and specificity of plasma analysis compared with tumor analysis to 83.3% and 100% (dPCR) and 57.1% and 100% (NGS), respectively. Among 25 patients with SPD, the T790M mutation of EGFR alone (n = 11), copy number gain (CNG) of NRAS (n = 1), CNG of MET (n = 1), CNG of EGFR plus T790M (n = 1), and CNG and E545K of PIK3CA plus T790M of EGFR (n = 1) were identified by NGS as putative resistance mechanisms against afatinib. No tumor showed transformation to small cell carcinoma. Median progression-free survival was longer in patients with than in those without T790M at SPD (15.1 versus 10.9 months, P =0.25). Median time to SPD was much longer in patients with than in those without T790M at SPD (17.9 versus 10.9 months, P =0.18). Assessment of T/A ratio with dPCR or NGS improved specificity of plasma analysis for determination of T790M positivity compared with tumor analysis. dPCR and NGS analysis in tumor and plasma samples shed light on exploring mechanisms of afatinib resistance.