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Background: Resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) has limited treatment options for patients with EGFR-mutated non-small-cell lung cancer (NSCLC). Although osimertinib or afatinib alone induced drug-resistant clones with EGFR secondary mutation in a preclinical model, its combination prevented the appearance of these mutations. We investigated alternating-dose therapy of osimertinib and afatinib in patients with EGFR-mutant NSCLC in a single-arm Phase II trial., Methods: Treatment-naïve patients with stage IV NSCLC harboring an activating EGFR mutation were enrolled. Alternating cycles of osimertinib (80 mg/day) followed by afatinib (20 mg/day) were administered every 8 weeks. Genomic analysis was performed using circulating tumor DNA obtained before and after the treatment., Results: Among the 46 enrolled patients, the median progression-free survival was 20.2 months. The overall response rate was 69.6%. The median overall survival was not reached. Among the 26 plasma samples obtained after the acquisition of resistance, 3 showed an increased MET gene copy number, and 1 showed BRAF mutation. Meanwhile, no EGFR secondary mutation was detected., Conclusion: The efficacy of our treatment was not significantly different from osimertinib alone, as reported previously in untreated advanced NSCLC patients with EGFR mutations. Although the sample size was limited, this treatment may prevent the emergence of EGFR secondary mutations that trigger drug resistance. Further studies are warranted to establish the significance of this treatment., Clinical Trial Registration: jRCTs051180009., Competing Interests: Disclosure The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Objective: Immune checkpoint inhibitors (ICIs) have become a key therapeutic modality for advanced non-small cell lung cancer (NSCLC), but most patients experience primary or acquired resistance to these drugs. We here explored the mechanisms underlying both types of ICI resistance by analysis of the tumor immune microenvironment (TME)., Materials and Methods: Four patients who experienced a long-term response to ICI treatment (progression-free survival [PFS] of ≥12 months) followed by disease progression, after which a rebiopsy was immediately performed (cohort-A), as well as four patients who experienced early tumor progression during ICI treatment (PFS of <9 weeks, cohort-B) were enrolled in this retrospective study. The pretreatment TME was evaluated by 16- or 17-color multiplex immunohistochemistry (mIHC)-based spatial profiling at the single-cell level for both cohorts. In cohort-A, changes in the TME after disease progression during ICI treatment were also investigated by mIHC analysis and transcriptomic analysis., Results: Pretreatment tumor tissue from cohort-B manifested poor infiltration of tumor-reactive CD8 + T cells characterized by CD39 and CD103 expression or by programmed cell death-1 expression, implicating insufficient recognition of tumor cells by CD8 + T cells as a mechanism of primary ICI resistance. Analysis of the paired tumor specimens from cohort-A revealed various changes in the TME associated with acquired ICI resistance, including substantial infiltration of myeloid-derived suppressor cells and M2-type tumor-associated macrophages without a marked decline in the number of tumor-reactive CD8 + T cells; a decrease in the number of tumor-reactive CD8 + T cells; and an apparent decrease in neoantigen presentation by tumor cells., Conclusion: The presence of intratumoral tumor-reactive CD8 + T cells may be a prerequisite for a long-term response to ICI treatment in advanced NSCLC, but it is not sufficient for cancer cell eradication. Various TME profiles are associated with acquired ICI resistance, suggesting that patient-specific strategies to overcome such resistance may be necessary., Competing Interests: Declaration of Competing Interest KH reports research funding regarding the submitted work from KANAE Foundation for the Promotion of Medical Science, SGH Foundation, and YOKOYAMA Foundation for Clinical Pharmacology; other research funding from AstraZeneca K.K. and MSD K.K.; honoraria from AS ONE Corporation, Bristol-Myers Squibb Company, MSD K.K., and Ono Pharmaceutical Co., Ltd., outside the submitted work; TT reports research funding from Ono Pharmaceutical Co., Ltd.; honoraria from Ono Pharmaceutical Co., Ltd., Merck Biopharma Co., Ltd., Merck Sharp & Dohme Corp, and Bristol Myers Squibb Co., Ltd., outside the submitted work; HK reports grants from Eisai Co., Ltd., Kobayashi Pharmaceutical. Co., Ltd., and Taiho Pharmaceutical Co., Ltd.; consulting fees from Daiichi-Sankyo Co., Ltd.; honoraria from Bristol-Myers Squibb Company, Eli Lilly Japan K.K., Ono Pharmaceutical Co., Ltd, Daiichi Sankyo Co., Ltd., Takeda Pharmaceutical Co., Ltd., Teijin Pharma Ltd, Otsuka Pharmaceutical Co., Ltd., Bayer Yakuhin Ltd., MSD K.K., Chugai Pharmaceutical Co., Ltd., Merck Biopharma Co., Ltd., Yakult Pharmaceutical Industry, and Taiho Pharmaceutical Co., Ltd., outside the submitted work; MT has received honoraria from Chugai Pharmaceutical Co., Ltd., AstraZeneca K.K., Bristol-Myers Squibb Company, Novartis Pharma K.K., Ono Pharmaceutical Co., Ltd., Boehringer Ingelheim Japan Inc., and Takeda Pharmaceutical Co., Ltd., outside the submitted work; KY reports research funding from Daiichi Sankyo Co., Ltd.; honoraria from AstraZeneca K.K., Chugai Pharmaceutical Co., Ltd., Eli Lilly Japan K.K., Takeda Pharmaceutical Co., Ltd., Merck Sharp & Dohme K.K., and Nippon Kayaku; patents US20200115467 and US20200061031 pending, outside the submitted work; KT has received honoraria from AstraZeneca K.K., Eisai Co., Ltd., Ono Pharmaceutical Co., Ltd., Chugai Pharmaceutical Co., Ltd., Taiho Pharmaceutical Co., Ltd., Merck Biopharma Co., Ltd., Bristol-Myers Squibb Company, MSD K.K., Takeda Pharmaceutical Co., Ltd., and Kyowa Hakko Kirin Co., Ltd., outside the submitted work; HH reports grants from AstraZeneca K.K., Astellas Pharma Inc., MSD K.K., Ono Pharmaceutical Co., Ltd., Nippon Boehringer Ingelheim Co., Ltd., Novartis Pharma K.K., Pfizer Japan Inc., Bristol Myers Squibb Co., Ltd., Eli Lilly Japan K.K., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Merck Serono Co., Ltd. / Merck Biopharma Co., Ltd., Takeda Pharmaceutical Co., Ltd., Taiho Pharmaceutical Co., Ltd., SymBio Pharmaceuticals Limited., AbbVie Inc, inVentiv Health Japan, ICON Japan K.K., GRITSONE ONCOLOGY.INC, PAREXEL International Corp., Kissei Pharmaceutical Co., Ltd., EPS Corporation., Syneos Health., Pfizer R&D Japan G.K., A2 Healthcare Corp., Quintiles Inc. / IQVIA Services JAPAN K.K., EP-CRSU CO., LTD., Linical Co., Ltd., Eisai Co., Ltd., CMIC Shift Zero K.K., Kyowa Hakko Kirin Co., Ltd, Bayer Yakuhin, Ltd, EPS International Co., Ltd., and Otsuka Pharmaceutical Co., Ltd.; honoraria from Amgen K.K., AstraZeneca K.K., Boehringer Ingelheim Japan Inc., Bristol-Myers Squibb Company, Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Eli Lilly Japan K.K., Guardant healthcare, Kyorin Pharmaceutical Co., Ltd., Merck Biopharma Co., Ltd., MSD K.K., Novartis Pharmaceuticals K.K., Ono Pharmaceutical Co., Ltd., Shanghai Haihe Biopharm, Taiho Pharmaceutical Co., Ltd., Pfizer Japan Inc., and Takeda Pharmaceutical Co., Ltd., outside the submitted work; KN reports grants from Boehringer Ingelheim Japan Inc., West Japan Oncology Group, Thoracic Oncology Research Group, North East Japan Study Group, Clinical Research Support Center Kyushu, Nichirei Biosciences Inc., and Eli Lilly Japan K.K.; consulting fees from SymBio Pharmaceuticals Ltd., Solasia Pharma, Eli Lilly Japan K.K., and Otsuka Pharmaceutical Co., Ltd.; honoraria from Boehringer Ingelheim Japan Inc., AstraZeneca K.K., Chugai Pharmaceutical Co., Ltd., Novartis Pharma K.K., Eisai Co., Ltd., MSD K.K., Bristol-Myers Squibb Company, Ono Pharmaceutical Co., Ltd., Pfizer Japan Inc., Sanofi K.K., Guardant Health, Inc., Eli Lilly Japan K.K., Amgen K.K., Merck Biopharma Co., Ltd., Roche Diagnostics K.K., Yakult Honsha Co., Ltd., Takeda Pharmaceutical Co., Ltd., Janssen Pharmaceutical K.K., FUJIREBIO Inc., outside the submitted work; KN reports grants from MSD K.K., ICON Japan K.K., Takeda Pharmaceutical Co., Ltd., Eli Lilly Japan K.K., Bristol-Myers Squibb Company, Taiho Pharmaceutical Co., Ltd., PAREXEL International Corp., Ono Pharmaceutical Co., Ltd., Sysmex Corporation, A2 Healthcare Corp., AbbVie Inc., Chugai Pharmaceutical Co., Ltd., Boehringer Ingelheim Japan Inc., SymBio Pharmaceuticals Limited., AstraZeneca K.K., EPS International Co., Ltd., CMIC Shift Zero K.K., Eisai Co., Ltd., Mochida Pharmaceutical Co., Ltd., Kissei Pharmaceutical Co., Ltd., GlaxoSmithKline K.K., Kyowa Hakko Kirin Co., Ltd., EPS Corporation Co., Ltd., Daiichi Sankyo Co., Ltd., Quintiles Inc. / IQVIA Services JAPAN K.K., Pfizer Japan Inc., Bayer Yakuhin Ltd., Otsuka Pharmaceutical Co., Ltd., PRA HEALTHSCIENCES, Merck Biopharma Co., Ltd., Covance Japan Inc., Medical Research Support, Sanofi K.K., Syneos Health., Pfizer R&D Japan G.K., PPD-SNBL K.K., and Japan Clinical Research Operations; consulting fees from Astellas Pharma Inc., Takeda Pharmaceutical Co., Ltd., Eli Lilly Japan K.K., Pfizer Japan Inc., KYORIN Pharmaceutical Co., Ltd., and Ono Pharmaceutical Co., Ltd.; honoraria from Astellas Pharma Inc., AstraZeneca K.K., MSD K.K., Ono Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Taiho Pharmaceutical Co., Ltd., Bristol Myers Squibb Co., Ltd., Medical Review Co., Ltd., Thermo Fisher Scientific K.K., KYORIN Pharmaceutical Co., Ltd., Nikkei Business Publications, Inc., Takeda Pharmaceutical Co., Ltd., Chugai Pharmaceutical Co., Ltd., Eli Lilly Japan K.K., Merck Biopharma Co., Ltd., Novartis Pharma K.K., Pfizer Japan Inc., CareNet, Inc, YODOSHA CO., LTD., Hisamitsu Pharmaceutical Co., Inc., MEDICUS SHUPPAN Publishers Co., Ltd., YOMIURI TELECASTING CORPORATION., NANZANDO Co., Ltd., Roche Diagnostics K.K., Nippon Kayaku Co., Ltd., Bayer Yakuhin, Co., Ltd., Kyowa Hakko Kirin Co., Ltd., AbbVie Inc., Amgen K.K., 3H Clinical Trial Inc., Nichi-Iko Pharmaceutical Co., Ltd., Nippon Boehringer Ingelheim Co., Ltd., and Medical Mobile Communications Co., Ltd., outside the submitted work. The remaining authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Introduction: Alternation of osimertinib and afatinib is a potential approach to overcome osimertinib resistance and to allow complementation of drug efficacy without compromising safety in patients with epidermal growth factor receptor gene (EGFR)-mutated non-small cell lung cancer (NSCLC)., Methods: Treatment-naive patients with stage IV NSCLC harboring an activating EGFR mutation (L858R or exon-19 deletion) were enrolled. Alternating cycles of osimertinib at 80 mg/day for 8 weeks followed by afatinib at 20 mg/day for 8 weeks were administered. The primary end point was 12-month progression-free survival (PFS) probability., Results: Forty-six patients were enrolled and treated with study therapy. The 12-month PFS probability was 70.2% (60% confidence interval [CI], 63.9-75.6%; 95% CI, 54.2-81.5%), which did not meet the primary end point. After a median follow-up time of 25.7 months, the median PFS was 21.3 months (95% CI, 16.3 months-not reached). The overall response rate was 69.6% (95% CI, 54.2-82.3%). The most common treatment-related adverse events (any grade or grade ≥ 3, respectively) were diarrhea (73.9%, 4.3%), rash acneiform (63.0%, 2.2%), and paronychia (52.2%, 0%). Five cases of pneumonitis, two of grade 2 and thres of grade 3, were apparent, all of which developed during osimertinib treatment. Exploratory evaluation of circulating tumor DNA suggested that coexisting TP53 mutations did not influence PFS for the alternating therapy., Conclusions: Alternating therapy with osimertinib and afatinib for treatment-naive patients with EGFR- mutated advanced NSCLC did not meet its primary end point, despite the encouraging efficacy and safety profile of this treatment strategy., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: EGFR-tyrosine kinase inhibitor (TKI) is a standard first-line therapy for activated EGFR -mutated non-small cell lung cancer (NSCLC). Treatment options for patients with acquired EGFR-TKI resistance are limited. HER3 mediates EGFR-TKI resistance. Clinical trials of the HER3-targeting antibody-drug conjugate patritumab deruxtecan (HER3-DXd) demonstrated its anticancer activity in EGFR -mutated NSCLC; however, the mechanisms that regulate HER3 expression are unknown. This study was conducted with the aim to clarify the mechanisms underlying HER3 regulation in EGFR -mutated NSCLC tumors and explored the strategy for enhancing the anticancer activity of HER3-DXd in EGFR -mutated NSCLC., Experimental Design: Paired tumor samples were obtained from 48 patients with EGFR -mutated NSCLC treated with EGFR-TKI(s). HER3 expression was immunohistochemically quantified with H-score, and genomic alteration and transcriptomic signature were tested in tumors from pretreatment to post-EGFR-TKI resistance acquisition. The anticancer efficacy of HER3-DXd and osimertinib was evaluated in EGFR -mutated NSCLC cells., Results: We showed augmented HER3 expression in EGFR -mutated tumors with acquired EGFR-TKI resistance compared with paired pretreatment samples. RNA sequencing revealed that repressed PI3K/AKT/mTOR signaling was associated with HER3 augmentation, especially in tumors from patients who received continuous EGFR-TKI therapy. An in vitro study also showed that EGFR-TKI increased HER3 expression, repressed AKT phosphorylation in multiple EGFR -mutated cancers, and enhanced the anticancer activity of HER3-DXd., Conclusions: Our findings help clarify the mechanisms of HER3 regulation in EGFR -mutated NSCLC tumors and highlight a rationale for combination therapy with HER3-DXd and EGFR-TKI in EGFR -mutated NSCLC., (©2021 American Association for Cancer Research.)

Purpose: Treatment with KRAS G12C inhibitors such as sotorasib can produce substantial regression of tumors in some patients with non-small cell lung cancer (NSCLC). These patients require alternative treatment after acquiring resistance to the inhibitor. The mechanisms underlying this acquired resistance are unclear. The purpose of this study was to identify the mechanisms underlying acquired sotorasib resistance, and to explore potential treatments for rescuing patients with sotorasib-resistant KRAS G12C NSCLC cells., Experimental Design: Clones of sotorasib-sensitive KRAS G12C NSCLC H23 cells exposed to different concentrations of sotorasib were examined using whole-genomic transcriptome analysis, multiple receptor kinase phosphorylation analysis, and gene copy-number evaluation. The underlying mechanisms of resistance were investigated using immunologic examination, and a treatment aimed at overcoming resistance was tested in vitro and in vivo ., Results: Unbiased screening detected subclonal evolution of MET amplification in KRAS G12C NSCLC cells that had developed resistance to sotorasib in vitro . MET knockdown using small interfering RNA (siRNA) restored susceptibility to sotorasib in these resistant cells. MET activation by its amplification reinforced RAS cycling from its inactive form to its active form. In addition to RAS-mediated MEK-ERK induction, MET induced AKT activation independently of RAS. Crizotinib, a MET inhibitor, restored sensitivity to sotorasib by eliminating RAS-MEK-ERK as well as AKT signaling. MET/ KRAS G12C dual inhibition led to tumor shrinkage in sotorasib-resistant xenograft mice., Conclusions: MET amplification leads to the development of resistance to KRAS G12C inhibitors in NSCLC. Dual blockade of MET and KRAS G12C could be a treatment option for MET -amplified, KRAS G12C -mutated NSCLC., (©2021 American Association for Cancer Research.)

Objectives: The T790M secondary mutation of epidermal growth factor receptor gene (EGFR) is the most common mechanism of acquired resistance to first- or second-generation EGFR tyrosine kinase inhibitors (TKIs). We investigated the association between gene mutation profile in EGFR mutation-positive non-small cell lung cancer (NSCLC) before EGFR-TKI treatment and T790M status after EGFR-TKI treatment., Materials and Methods: A total of 57 EGFR mutation-positive NSCLC patients who had undergone a repeat biopsy (tissue or liquid) after failure of treatment with a first- or second-generation EGFR-TKI and who had sufficient tumor tissue available from before treatment for genetic analysis was enrolled. The gene mutation profile of tumor tissue obtained before EGFR-TKI treatment was evaluated by next-generation sequencing with a comprehensive cancer gene panel (409 genes). The number of potentially damaging nonsynonymous mutations was predicted with PolyPhen-2 software., Results: Progression-free survival during EGFR-TKI treatment did not differ significantly between patients who developed T790M-mediated resistance and those who developed T790M-independent resistance. The predicted number of damaging nonsynonymous mutations in pretreatment tumor tissue was significantly lower in patients who developed T790M-mediated resistance than in those with T790M-independent resistance (P =  0.049)., Conclusions: Coexisting mutations in tumor tissue before EGFR-TKI treatment may contribute to the emergence of cell clones responsible for development of T790M-dependent or T790M-independent TKI resistance in patients with EGFR-mutated NSCLC. Multiplex genomic testing of pretreatment tumor tissue may thus provide a means of identifying patients likely to develop T790M-mediated TKI resistance and therefore inform treatment selection., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are standard therapy for EGFR-mutant non-small cell lung cancer (NSCLC). Preclinically, HER3 ligand heregulin induces resistance to EGFR-TKIs, whereas the pan-human EGFR family inhibitor afatinib remains effective. Here, we examined whether soluble heregulin levels have clinical implications for EGFR-mutant NSCLC treated with EGFR-TKIs. Soluble heregulin was immunologically measured in plasma from EGFR-mutant NSCLC patients. Cutoff values were determined by 1-year PFS ROC curve. The relationship between soluble heregulin and PFS following EGFR-TKI therapy was analyzed by Cox proportional hazards model. Seventy-three patients were enrolled: 44 were treated with 1 st -generation and 29 with 2 nd -generation EGFR-TKIs. Soluble heregulin levels varied (range: 274-7,138 pg/mL, median: 739 pg/mL). Among patients treated with 1 st -generation EGFR-TKIs, those with high heregulin (n = 20, >800 pg/mL) had a tendency for shorter PFS than those with low heregulin (n = 24, <800 pg/mL), with median PFS of 322 and 671 days, respectively. Cox proportional hazards model also indicated a trend toward resistance against 1 st -generation EGFR-TKIs (HR: 1.825, 95% CI: 0.865-4.318) but not against 2 nd -generation EGFR-TKIs. Soluble heregulin potentially correlates with resistance to EGFR-TKIs but not 2 nd -generation EGFR-TKIs in patients with EGFR-mutant NSCLC.

Background: Non-small cell lung cancer (NSCLC) positive for activating mutations of the epidermal growth factor receptor (EGFR) gene is initially sensitive to EGFR-tyrosine kinase inhibitors (TKIs) but eventually develops resistance to these drugs. Upregulation of the receptor tyrosine kinase AXL in tumor tissue has been detected in about one-fifth of NSCLC patients with acquired resistance to EGFR-TKIs. However, the clinical relevance of the levels of AXL and its ligand GAS6 in plasma remains unknown., Methods: Tumor tissue and plasma specimens were collected from 25 EGFR-mutated NSCLC patients before EGFR-TKI treatment or after treatment failure. The levels of AXL and of GAS6 mRNA in tumor tissue were evaluated by immunohistochemistry and chromogenic in situ hybridization, respectively. The plasma concentrations of AXL and GAS6 were measured with enzyme-linked immunosorbent assays., Results: AXL expression was detected in three of 12 (25%) and nine of 19 (47%) tumor specimens obtained before and after EGFR-TKI treatment, respectively. All tumor specimens assayed were positive for GAS6 mRNA. The median values for the plasma AXL concentration before and after EGFR TKI treatment were 1 635 and 1 460 pg/mL, respectively, and those for the plasma GAS6 concentration were 4 615 and 6 390 pg./mL, respectively. There was no significant correlation between the plasma levels of AXL or GAS6 and the corresponding expression levels in tumor tissue., Conclusion: Plasma concentrations of AXL and GAS6 do not reflect tumor expression levels, and their measurement is thus not a viable alternative to direct analysis of tumor tissue in EGFR-mutated NSCLC., (© 2019 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR‑TKIs) are efficacious drugs for non‑small cell lung cancers (NSCLCs) with EGFR‑activating mutations. Afatinib, a second‑generation EGFR‑TKI and osimertinib, a third‑generation EGFR‑TKI, are both standard therapies for patients with these types of cancer. Each drug possesses distinct binding sites for the tyrosine kinase domain of EGFR. The present study examined the efficacy of single and combination TKI therapy using in vitro growth inhibition assays of Ba/F3 cells with an EGFR‑activating Del19 mutation. Afatinib or osimertinib treatment alone markedly inhibited cell proliferation in Ba/F3 cells, although drug‑resistant cells eventually appeared with secondary EGFR mutations (either T790M or C797S, respectively) as determined by direct sequencing. Notably a combination of afatinib and osimertinib eradicated Ba/F3 cells with no development of resistance. We also evaluated the efficacy of afatinib, osimertinib, and a combination of the two, using drug‑resistant cells with T790M or C797S mutations. Osimertinib was effective for treating Ba/F3 cells with the T790M mutation, whereas afatinib was moderately effective against C797S Ba/F3 cells. However, subsequent treatment, even when both drugs were used in combination, could not completely eradicate the Ba/F3 population and doubly resistant cells with a variety of triple mutations were generated, including Del19/T790M/C797S. In conclusion, an initial treatment with a combination of osimertinib and afatinib is potentially more effective for eradicating mutant EGFR‑dependent cells than sequential drug use. This should be tested in future clinical trials to establish whether such a combination would be effective for the treatment of NSCLC.

EGFR tyrosine kinase inhibitors (TKIs) are standard therapy for EGFR-mutant non-small cell lung cancer (NSCLC); however, these tumours eventually acquire chemoresistance. U3-1402 is an anti-HER3 antibody-drug conjugate with a novel topoisomerase I inhibitor, DXd. In the current study, we evaluated the anticancer efficacy of U3-1402 in EGFR-mutant NSCLC cells with acquired resistance to EGFR-TKIs. HCC827GR5 and PC9AZDR7 are EGFR-TKI-resistant clones for gefitinib and osimertinib, respectively. U3-1402 alone or in combination with the EGFR-TKI erlotinib demonstrated potent anticancer efficacy in HCC827GR5 cells using an in vitro growth inhibition assay and in vivo xenograft mouse model. U3-1402 induced apoptosis in HCC827GR5 cells accompanying phosphorylation of histone H2A.X, a marker of DNA damage, but did not block HER3/PI3K/AKT signalling. Further, we found using flow cytometry that the cell surface HER3 expression level in HCC827GR5 cells was twice that found in HCC827 cells, indicating internalization of U3-1402 was increased in resistant cells. In addition, administration of U3-1402 notably repressed growth of EGFR-TKI osimertinib-resistant PC9AZDR7 xenograft tumours, and that PC9AZDR7 cells expressed five times greater cell surface HER3 than PC9 cells. Furthermore, using immunofluorescent microscopy, HER3 was observed predominantly in the nucleus of PC9 cells, but was localized in the cytoplasm of PC9AZDR7 cells. This finding indicates that altered trafficking of the HER3-U3-1402 complex may accelerate linker payload cleavage by cytoplasmic lysosomal enzymes, resulting in DNA damage. Our results indicate that administration of U3-1402 alone or in combination with an EGFR-TKI may have potential as a novel therapy for EGFR-TKI-resistant EGFR-mutant NSCLC.

The efficacy of programmed cell death-1 (PD-1) blockade in patients with non-small cell lung cancer (NSCLC) positive for epidermal growth factor receptor (EGFR) gene mutations has been found to be limited, but the underlying mechanisms for this poor response have remained obscure. Given that the recognition by T cells of tumor antigens presented by major histocompatibility complex class I (MHC-I) molecules is essential for an antitumor immune response, we examined the effects of EGFR tyrosine kinase inhibitors (TKIs) on MHC-I expression in NSCLC cell lines. Appropriate EGFR-TKIs increased MHC-I expression at the mRNA and cell surface protein levels in NSCLC cells positive for EGFR mutations including those with the T790M secondary mutation. Trametinib, an inhibitor of the extracellular signal-regulated kinase (ERK) kinase MEK, also increased MHC-I expression, whereas the phosphatidylinositol 3-kinase (PI3K) inhibitor buparlisib did not, suggesting that the MEK-ERK pathway mediates the down-regulation of MHC-I expression in response to EGFR activation. Immunohistochemical analysis of EGFR-mutated NSCLC specimens obtained before and after EGFR-TKI treatment also revealed down-regulation of phosphorylated forms of EGFR and ERK in association with up-regulation of MHC-I, an increased number of infiltrating CD8 + T cells, and increased PD-1 ligand 1 expression after such treatment. Our results thus suggest that mutational activation of EGFR inhibits MHC-I expression through the MEK-ERK pathway in NSCLC and thereby contributes to the poor response of such tumors to immunotherapy. Further studies are warranted to evaluate the relation between EGFR-MEK-ERK signaling in and the immune response to EGFR-mutated NSCLC. ., (© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Importance: Immune-related adverse events (irAEs) have been associated with the efficacy of PD-1 (programmed cell death protein 1) inhibitors in patients with melanoma, but whether such an association exists for non-small-cell lung cancer (NSCLC) has remained unknown., Objective: To evaluate the relation of irAEs to nivolumab efficacy in NSCLC., Design, Setting, and Participants: In this study based on landmark and multivariable analyses, a total of 134 patients with advanced or recurrent NSCLC who were treated with nivolumab in the second-line setting or later between December 2015 and August 2016 were identified from a review of medical records from multiple institutions, including a university hospital and community hospitals. Data were updated as of December 31, 2016., Exposures: The absence or presence of any irAE before the landmark date., Main Outcomes and Measures: Kaplan-Meier curves of progression-free survival (PFS) according to the development of irAEs in 6-week landmark analysis were evaluated with the log-rank test as a preplanned primary objective. Overall survival (OS) was similarly evaluated. Multivariable analysis of both PFS and OS was performed with Cox proportional hazard regression models., Results: In a cohort of 134 patients (median [range] age, 68 [33-85] years; 90 men [67%], 44 women [33%]), irAEs were observed in 69 of the 134 study patients (51%), including 12 patients (9%) with such events of grade 3 or 4, and 24 patients (18%) requiring systemic corticosteroid therapy. In 6-week landmark analysis, median PFS was 9.2 months (95% CI, 4.4 to not reached [NR]) and 4.8 months (95% CI, 3.0 to 7.5) (P = .04) whereas median OS was NR (95% CI, 12.3 to NR) and 11.1 months (95% CI, 9.6 to NR) (P = .01) for patients with or without irAEs, respectively. Multivariable analysis also revealed that irAEs were positively associated with survival outcome, with hazard ratios of 0.525 (95% CI, 0.287 to 0.937; P = .03) for PFS and 0.282 (95% CI, 0.101 to 0.667; P = .003) for OS., Conclusions and Relevance: Development of irAEs was associated with survival outcome of nivolumab treatment in patients with advanced or recurrent NSCLC. Further studies are needed to confirm our findings.

Objective: The aim of this study was to identify baseline peripheral blood biomarkers associated with clinical outcome in patients with NSCLC treated with nivolumab., Methods: Univariable and multivariable analyses were performed retrospectively for 134 patients with advanced or recurrent NSCLC treated with nivolumab to evaluate the relationship between survival and peripheral blood parameters measured before treatment initiation, including absolute neutrophil count (ANC), absolute lymphocyte count (ALC), absolute monocyte count, and absolute eosinophil count (AEC), as well as serum C-reactive protein and lactate dehydrogenase levels. Progression-free survival, overall survival, and response rate were determined., Results: Among the variables selected by univariable analysis, a low ANC, high ALC, and high AEC were significantly and independently associated with both better progression-free survival (p = 0.001, p = 0.04, and p = 0.02, respectively) and better overall survival (p = 0.03, p = 0.03, and p = 0.003, respectively) in multivariable analysis. Categorization of patients according to the number of favorable factors revealed that those with only one factor had a significantly worse outcome than those with two or three factors. A similar trend was apparent for patients with a programmed death 1 ligand tumor proportion score less than 50%, whereas all patients with a score of 50% or higher had at least two favorable factors., Conclusions: A baseline signature of a low ANC, high ALC, and high AEC was associated with a better outcome of nivolumab treatment, with the number of favorable factors identifying subgroups of patients differing in survival and response rate., (Copyright © 2017 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

T790M mutation-selective EGFR tyrosine kinase inhibitors (EGFR-TKI) have demonstrated clinical benefits in non-small cell lung cancer (NSCLC) patients harboring T790M mutation, which is the major cause of resistance to EGFR-TKI. However, their efficacy is limited, possibly due to the emergence of apoptosis resistance in T790M-positive NSCLC. We previously identified Src family kinases as cooncogenic drivers along with T790M and found that the Src inhibitor dasatinib combined with an irreversible or a preclinical T790M-selective EGFR-TKI enhanced antitumor activity in T790M-positive cells. In the current study, we evaluated the efficacy of dasatinib combined with the clinically relevant T790M-selective EGFR-TKI ASP8273 or osimertinib in EGFR mutation-positive NSCLC with or without T790M mutation. A cell viability assay revealed that dasatinib had synergistic effects with these TKIs in T790M-positive cells and simultaneously inhibited Src, Akt, and Erk, which remained activated upon single-agent treatment. Dasatinib also increased the rate of apoptosis in T790M-positive cells induced by T790M-selective EGFR-TKIs, as determined by the Annexin-V binding assay; this was associated with downregulation of the antiapoptotic Bcl-2 family member Bcl-xL, a finding that was confirmed in mice bearing T790M-positive xenografts. Our results suggest that Bcl-xL plays a key role in the apoptosis resistance of T790M-positive NSCLC, and that dasatinib combined with clinically relevant T790M-selective EGFR-TKIs is potentially effective in overcoming resistance to first-generation EGFR-TKIs in NSCLC patients with acquired T790M. Mol Cancer Ther; 16(11); 2563-71. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Background: This phase 1 study evaluated the safety, tolerability, pharmacokinetics and efficacy of patritumab (U3-1287) Process 2, a new formulation of fully human anti-HER3 monoclonal antibody in combination with erlotinib, an epidermal growth factor receptortyrosine kinase inhibitor (EGFR-TKI) in prior chemotherapy treated Japanese patients with advanced non-small cell lung cancer (NSCLC)., Methods: Patients received intravenous patritumab Process 2 formulation at 9 mg/kg every 3 weeks after initiation of 18 mg/kg loading dose combined with continuous daily dose of erlotinib (150 mg QD) until any of the withdrawal criteria are met. Adverse events (AEs) were assessed using CTCAE v4.0 and tumor response was assessed using RECIST v1.1. Full pharmacokinetic sampling and serum biomarker analyses were mainly performed during cycle 1 and 2., Results: Total of six EGFR-mutant NSCLC patients including one EGFR-TKI naïve patient received patritumab Process 2 formulation combined with erlotinib. No dose-limiting toxicities were observed. The most frequent AEs were gastrointestinal or skin toxicities, which were generally mild and manageable. One patient discontinued from study due to reversible grade 3 interstitial lung disease. The mean area under the curve (AUC) value was 2640 μg/day/mL; the Cmax value was 434 μg/mL, respectively. The median progression-free survival (95% confidence interval) was 220.0 (100.0-363.0) days. HER3 ligand heregulin was detected in serum from only a patient that maintained most durable stable disease., Conclusions: Patritumab Process 2 formulation in combination with erlotinib was well tolerated compatible with favorable PK profile in Japanese patients with advanced NSCLC.

Objectives: Patritumab is a fully human anti-human epidermal growth factor receptor 3 (HER3) antibody that blocks activation by its ligand, heregulin (HRG). Preclinical studies have demonstrated the efficacy of patritumab in aberrantly high HRG-expressing non-small cell lung cancer (NSCLC). In the phase II randomized, placebo-controlled double-blind study HERALD (n=212 patients with NSCLC), patritumab plus erlotinib did not improve progression-free survival (PFS) compared with placebo plus erlotinib. The current study examined whether soluble HRG (sHRG) level in serum correlated with the efficacy of patritumab plus erlotinib., Materials and Methods: Serum was obtained from participants prior to treatment (n=202). sHRG level was measured using a validated quantitative immune assay, and correlations with survival were blindly assessed., Results: sHRG level was various (-1346-11,772pg/mL). Participants were divided into the sHRG-high or -low subgroups at the concentration defining near the third quartile, 980pg/mL. Patritumab plus erlotinib significantly improved PFS relative to placebo in the sHRG-high subgroup (n=46, hazard ratio 0.42 [0.19-0.96], p=0.0327). In contrast, the HRG-low subgroup (n=148) had no improvement in PFS with patritumab., Conclusion: sHRG seems to be a predictive biomarker for the efficacy of patritumab plus erlotinib in NSCLC patients., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Bevacizumab combined with platinum-based chemotherapy has been established as a standard treatment option in the first-line setting for advanced nonsquamous non-small cell lung cancer (NSCLC). However, there has been no evidence to support the use of bevacizumab beyond disease progression in such patients., Methods: West Japan Oncology Group 5910L was designed as a multicenter, open-label, randomized, phase 2 trial of docetaxel versus docetaxel plus bevacizumab every 3 weeks for patients with recurrent or metastatic nonsquamous NSCLC whose disease had progressed after first-line treatment with bevacizumab plus a platinum-based doublet. The primary endpoint was progression-free survival (PFS)., Results: One hundred patients were randomly assigned to receive docetaxel (n = 50) or docetaxel plus bevacizumab (n = 50), and this yielded median PFS times of 3.4 and 4.4 months, respectively, with a hazard ratio (HR) of 0.71 and a stratified log-rank P value of .058, which met the predefined criterion for statistical significance (P < .2). The median overall survival also tended to be longer in the docetaxel plus bevacizumab group (13.1 months; 95% confidence interval [CI], 10.6-21.4 months) versus the docetaxel group (11.0 months; 95% CI, 7.6-16.1 months) with an HR of 0.74 (95% CI, 0.46-1.19; stratified log-rank P = .11). No unexpected or severe adverse events were recorded., Conclusions: Further evaluation of bevacizumab beyond disease progression is warranted for patients with advanced NSCLC whose disease has progressed after treatment with bevacizumab plus a platinum-based doublet., (© 2016 American Cancer Society.)

Afatinib is a second generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) characterized as an irreversible pan-human EGFR (HER) family inhibitor. Afatinib remains effective for a subpopulation of patients with non-small cell lung cancer (NSCLC) with acquired resistance to first generation EGFF-TKIs such as erlotinib. Heregulin activates HER3 in an autocrine fashion and causes erlotinib resistance in NSCLC. Here we examine whether afatinib is effective against heregulin-overexpressing NSCLCs harboring EGFR activating mutations. Afatinib but not erlotinib decreased EGFR mutant NSCLC PC9HRG cell proliferation in vitro and in mouse xenografts. Afatinib inhibited phosphorylation of the cell signaling pathway proteins HER3, EGFR, HER2, and HER4, likely by prevention of trans-phosphorylation as HER3 kinase activity is inadequate for auto-phosphorylation. Afatinib, unlike erlotinib, inhibited AKT activation, resulting in elevated apoptosis in PC9HRG cells. Clinically, a subpopulation of 33 patients with EGFR mutations and NSCLC who had received first generation EGFR-TKIs exhibited elevated plasma heregulin levels compared to healthy volunteers; one of these achieved a response with afatinib therapy despite having previously developed erlotinib resistance. Afatinib can overcome heregulin-mediated resistance to erlotinib in EGFR mutant NSCLC. Further studies are necessary to determine whether heregulin can predict afatinib efficacy after development offirst generation EGFR-TKI resistance.

Aim: To evaluate the tolerability and efficacy of erlotinib treatment in advanced non-small cell lung cancer (NSCLC) patients who had previously experienced severe hepatotoxicity after gefitinib treatment., Patients and Methods: Twenty-five NSCLC patients with epidermal growth factor receptor (EGFR) mutation were initially treated with gefitinib (250 mg/day). However, 7 of these experienced severe hepatotoxicity. After recovery from hepatotoxicity, treatment was switched to erlotinib (150 mg/day) in all 7 patients. Toxicity and efficacy of erlotinib were analyzed., Results: None of the 7 patients reported severe hepatotoxicity with erlotinib despite gefitinib-induced severe hepatotoxicity. All patients achieved response with gefitinib or following erlotinib treatment. The response achieved with gefitinib was maintained after switching to erlotinib. Therefore, an excellent median progression-free survival of 372 days was achieved although gefitinib induced severe hepatotoxicity., Conclusion: Erlotinib treatment was efficient and well-tolerated in NSCLC patients with EGFR mutation, despite their severe hepatotoxicity with prior gefitinib treatment., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

A 72-year-old woman with dysphagia was diagnosed with lung adenocarcinoma and metastatic meningeal tumour that impaired the medulla. Owing to a bulky tumour beside the medulla, radiosurgical control of the meningeal tumour was achieved before systemic therapy. Genomic examination of the tumour revealed an existing epidermal growth factor receptor (EGFR) exon 19 deletion, for which an EGFR tyrosine kinase inhibitor such as gefitinib was the standard therapy. However, because of dysphagia, the patient was unable to orally ingest gefitinib. Gefitinib was delivered via gastrostomy tube as a suspension after spontaneous dissolution in hot water. One month later, the patient's symptoms, including dysphagia, were drastically improved and she had recovered sufficiently to orally ingest gefitinib. Gefitinib-associated toxicity comprises only mild liver dysfunction and skin rash. CT scanning and MRI detected drastic shrinkage of the primary lung and meningeal tumours. The patient continued to take gefitinib and has remained symptom-free for 9 months.

Background: A phase I study was performed to evaluate dose-limiting toxicity and the recommended dose for the oral fluoropyrimidine S-1 administered concurrently with thoracic radiotherapy (TRT) in elderly (≥ 70 years of age) patients with locally advanced non-small cell lung cancer., Methods: S-1 was administered on days 1 to 14 and 22 to 35 at oral doses of 65 or 80 mg m(-2) day(-1). TRT was administered in 2-Gy fractions five times weekly for a total dose of 60 Gy. Twelve previously untreated patients were treated with S-1 at 65 (n=6) or 80 (n=6) mg m(-2) day(-1)., Results: All patients completed the planned 60 Gy of TRT. Dose-limiting toxicity included pneumonitis (n=2), infection (n=1), and stomatitis (n=1), each of grade 3, but each event was reversible. The recommended dose for S-1 was determined to be 80 mg m(-2) day(-1). No patient experienced toxicity of grade 4. The dose intensity of S-1 was well maintained and the combination of S-1 plus TRT was well tolerated overall. The overall response rate was 83.3 %, with a median survival time of 34.0 months., Conclusions: Administration of S-1 at 80 mg m(-2) day(-1) on days 1 to 14 and 22 to 35 can be safely combined with concurrent TRT in elderly patients with locally advanced non-small cell lung cancer.

Introduction: The primary objectives of this study were to determine the recommended dose of pemetrexed and carboplatin in patients with chemo-naive advanced non-small cell lung cancer (NSCLC)., Methods: Patients received escalated doses of carboplatin area under the concentration-time curve (AUC) of 5 (cohort 1) or 6 (cohort 2) and pemetrexed 500 mg/m(2) every 3 weeks for six cycles. For patients with objective response and stable disease, pemetrexed were continued until disease progression or unacceptable toxicity., Results: In cohort 1, a dose-limiting toxicity (DLT) was observed in one of the six patients: grade 4 thrombocytopenia. No DLTs were seen in the first 6 patients of cohort 2, and thus the combination of pemetrexed 500 mg/m(2) plus carboplatin at AUC 6 was determined as the recommended dose. Among a total of 20 patients, 8 patients received a median of four cycles of pemetrexed monotherapy in a maintenance setting without unexpected or cumulative toxicities. No complete responses and 12 partial responses were observed, giving an overall response rate of 60.0% [95% confidence interval (CI), 36.1-80.9%]. Median progression-free survival time for all patients was 7.6 months (95% CI: 4.8-8.0 months)., Conclusions: Pemetrexed 500 mg/m(2) plus carboplatin AUC 6 combination therapy followed by pemetrexed maintenance therapy, is generally tolerable, and shows encouraging antitumor activity in chemotherapy-naive patients with advanced NSCLC., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Sorafenib is a multikinase inhibitor whose targets include B-RAF and C-RAF, both of which function in the extracellular signal-regulated kinase (ERK) signaling pathway but which also have distinct downstream targets. The relative effects of sorafenib on B-RAF and C-RAF signaling in tumor cells remain unclear, however. We have now examined the effects of sorafenib as well as of B-RAF or C-RAF depletion by RNA interference on cell growth and ERK signaling in non-small cell lung cancer (NSCLC) cell lines with or without KRAS mutations. Sorafenib inhibited ERK phosphorylation in cells with wild-type KRAS but not in those with mutant KRAS. Despite this difference, sorafenib inhibited cell growth and induced G(1) arrest in both cell types. Depletion of B-RAF, but not that of C-RAF, inhibited ERK phosphorylation as well as suppressed cell growth and induced G(1) arrest in cells with wild-type KRAS. In contrast, depletion of C-RAF inhibited cell growth and induced G(1) arrest, without affecting ERK phosphorylation, in cells with mutant KRAS; depletion of B-RAF did not induce G(1) arrest in these cells. These data suggest that B-RAF-ERK signaling and C-RAF signaling play the dominant roles in regulation of cell growth in NSCLC cells with wild-type or mutant KRAS, respectively. The G(1) arrest induced by either C-RAF depletion or sorafenib in cells with mutant KRAS was associated with down-regulation of cyclin E. Our results thus suggest that sorafenib inhibits NSCLC cell growth by targeting B-RAF in cells with wild-type KRAS and C-RAF in those with mutant KRAS.

Purpose: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, gefitinib and erlotinib, lead to significant tumor regressions in 10% to 15% of non-small cell lung cancer (NSCLC) patients with EGFR activating mutations. However, 30% to 40% of NSCLC patients, majority of whom are EGFR wild-type, develop stable disease following EGFR tyrosine kinase inhibitor therapy. EGFR-directed antibodies (cetuximab) are effective treatments for head and neck squamous cell carcinomas, which seldom contain EGFR mutations. The determinant(s) of efficacy of EGFR-targeted therapies in EGFR wild-type cancers is not well defined., Experimental Design: We examined the relationship of EGFR ligands, EGF, transforming growth factor-alpha,and amphiregulin and the efficacy of gefitinib and cetuximab in EGFR wild-type NSCLC (n=10) and head and neck squamous cell carcinoma (n=4) cell lines. We compared amphiregulin expression using immunohistochemistry in EGFR wild-type NSCLC patients (n=24) that developed either stable or progressive disease following erlotinib or gefitinib treatment., Results: Cell lines which produced >or=20 pmol/L amphiregulin, as detected by an ELISA, were significantly more likely to be growth inhibited by both gefitinib and cetuximab than those that produced minimal or no amphiregulin. In these cell lines, both cetuximab and gefitinib led to cell cycle arrest at the G(1)-S boundary and was associated with preferential inhibition of extracellular signal-regulated kinase 1/2 but not Akt signaling. Amphiregulin expression was significantly higher in NSCLC patients that developed stable disease compared with those that developed disease progression following gefitinib or erlotinib treatment., Conclusions: Amphiregulin expression may help select EGFR wild-type patients who are likely to develop stable disease from EGFR-targeted therapies.