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Background: The aim of this study was to evaluate the potential of liquid biopsy for prediction of the efficacy of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) treatment and for assessment of the changes in genetic alterations during such treatment.Methods: Plasma samples were prospectively collected from non-small cell lung cancer patients with EGFR-activating mutations during EGFR-TKI treatment until disease progression and were analyzed for such mutations with droplet digital polymerase chain reaction and for other somatic alterations with next-generation sequencing.Results: One hundred patients, including 87 who were EGFR-TKI naïve, were enrolled. Median progression-free survival was significantly shorter for EGFR-TKI-naïve patients with EGFR-activating mutations detected in plasma at baseline than for those without them (7.9 vs 19.0 months; P < .001), with the values being significantly longer for initially positive patients who became negative for these mutations at 12 or 24 weeks than for those who remained positive. An increase in the number of alleles positive for EGFR-activating mutations in plasma during treatment was associated with disease progression, with a hazard ratio of 4.72 (95% CI, 2.07-10.79; P < .001) for EGFR-TKI-naïve patients showing an increase within 36 weeks. For 55 patients with available samples, the total number of somatic alterations (other than activating mutations or T790M of EGFR) in plasma was higher at disease progression than at baseline (33 vs 19; P = 0.04).Conclusion: Liquid biopsy shows potential for prediction of EGFR-TKI efficacy and elucidation of clonal tumor evolution during targeted therapy. [ABSTRACT FROM AUTHOR]

[Display omitted] • Robo2 can bind to NELL1/2 in acidic conditions but not physiological conditions. • The occluded NELL1/2-binding site is exposed by conformational change of Robo2. • Alternative splicing isoforms of Robo2 have distinct NELL1/2-binding affinities. • FRET-based indicators reveal that Robo2 changes conformation depending on acidic pH. Since neural epidermal growth factor-like-like (NELL) 2 was identified as a novel ligand for the roundabout (Robo) 3 receptor, research on NELL–Robo signaling has become increasingly important. We have previously reported that Robo2 can bind to NELL1/2 in acidic conditions but not at neutral pH. The NELL1/2-binding site that is occluded in neutral conditions is thought to be exposed by a conformational change of the Robo2 ectodomain upon exposure to acidic pH; however, the underlying structural mechanisms are not well understood. Here, we investigated the interaction between the immunoglobulin-like domains and fibronectin type III domains that form hairpin-like structure of the Robo2 ectodomain, and demonstrated that acidic pH attenuates the interaction between them. Alternative splicing isoforms of Robo2, which affect the conformation of the hairpin-like structure, were found to have distinct NELL1/2-binding affinities. We developed Förster resonance energy transfer-based indicators for monitoring conformational change of the Robo2 ectodomain by individually inserting donor and acceptor fluorescent proteins at its ends. These experiments revealed that the ends of the Robo2 ectodomain are close to each other in acidic conditions. By combining these findings with the results of size exclusion chromatography analysis, we suggest that, in acidic conditions, the Robo2 ectodomain has a compact conformation with a loose hairpin-like structure. These results may help elucidate the signaling mechanisms resulting from the interaction between Robo2 and NELL1/2 in acidic conditions. [ABSTRACT FROM AUTHOR]

Objectives. To evaluate the efficacy and safety of personalized peptide vaccination (PPV) combined with chemotherapy for patients with previously treated advanced non-small-cell lung cancer (NSCLC). Patients and Methods. Previously treated PS0-1 patients with IIIB/IV EGFR (epidermal growth factor receptor) wild genotype NSCLC were randomly assigned to docetaxel (60 mg/m(2) on Day 1) plus PPV based on preexisting host immunity or docetaxel plus placebo. Docetaxel administration was repeated every 3 weeks until disease progression. Personalized peptides or placebo was injected subcutaneously weekly in the first 8 weeks and biweekly in subsequent 16 weeks. The primary efficacy endpoint was progression-free survival (PFS). Results. PPV related toxicity was grade 2 or less skin reaction. The median PFS for placebo arm and PPV arm was 52 days and 59 days, respectively. There was no significant difference between two arms by log-rank test (p = 0.42). Interestingly, PFS and overall survival (OS) in humoral immunological responder were significantly longer than those in nonresponder. Conclusion. PPV did not improve the survival in combination with docetaxel for previously treated advanced NSCLC. However, PPV may be efficacious for the humoral immunological responders and a further clinical investigation is needed. [ABSTRACT FROM AUTHOR]

The genetic backgrounds of the Japanese (or Asians) are, at least in part, different from those of Caucasians. It is necessary to recognize this difference to develop medicine that is both optimized and individualized. In particular, the consideration of ethnic differences is becoming increasingly important for lung cancer medicine. Japanese clinical practice guidelines indicate that some clinical biomarkers, such as epidermal growth factor receptor gene mutations, echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase fusion gene and uridine diphosphate glucuronosyltransferase genotypes should be determined in appropriate lung cancer patients. At the present time, tests for these biomarkers are covered by the Japanese national health-care programme, as is treatment with certain targeted drugs and cytotoxic agents. Therefore, most patients with lung cancer in Japan receive these tests as part of daily practice if their performance status and organ function are judged to be eligible. In addition, ethnic differences in bone marrow toxicity caused by cytotoxic drugs are reflected in treatment choice, and the requirements for the development of treatment modalities suitable for rare targeted populations are also increasing. To meet these requirements, many collaborative groups in Japan that have improved their infrastructure for investigator-initiated trials and conducted important activities need to provide further optimal treatment modalities for Japanese and Asian patients with lung cancer. Here, the characteristics of lung cancer in Japanese patients, general aspects of medical treatment and the care system in Japan, and representative studies on lung cancer in Japan are reviewed. [ABSTRACT FROM AUTHOR]

• HER2 forms heterodimers with EGFR in cell lines without HER2 amplification. • HER2-EGFR heterodimers are abundant in cell lines with EGFR mutations. • HER2 is rapidly internalized as a result of its heterodimerization with EGFR. • HER2 is efficiently transferred to lysosomes in cells with EGFR mutations. • T-DM1 shows high cytotoxic efficacy in cells with EGFR mutations. Human epidermal growth factor receptor 2 (HER2) forms homodimers and is retained at the surface of cancer cells positive for HER2 amplification. The dimerization, internalization, and intracellular trafficking of HER2 in cancer cells without HER2 amplification have remained uncharacterized, however. HER2 homodimers and heterodimers were detected in various cell lines with the use of an in situ proximity ligation assay. The effects of wild-type or mutant forms of epidermal growth factor receptor (EGFR) on intracellular trafficking of HER2 were examined by live-cell imaging. The sensitivity of cell lines without HER2 amplification to ado-trastuzumab emtansine (T-DM1), an anti-HER2 (trastuzumab)–cytotoxic drug conjugate (ADC) was also investigated. HER2 preferentially formed heterodimers with EGFR rather than homodimers and was rapidly internalized together with EGFR in cells without HER2 amplification. HER2-EGFR heterodimers were more abundant and HER2 was more efficiently transferred to lysosomes in such cells with than in those without EGFR activating mutations. T-DM1 showed a high cytotoxic efficacy in the cells with EGFR mutations, suggesting that mutant forms of EGFR promote the transfer of HER2-bound T-DM1 to lysosomes through efficient formation of HER2-EGFR heterodimers. Our findings reveal that HER2 trafficking is affected by EGFR, especially by mutant forms of the receptor, and they provide a rationale for the use of HER2-targeting ADCs in the treatment of EGFR -mutated lung cancer. [ABSTRACT FROM AUTHOR]

Human epidermal growth factor receptor 2 (HER2) mutations are present in ∼3% of patients with non–small cell lung cancer (NSCLC), with exon-20 insertions accounting for ∼90% of such HER2 mutations and having been identified as driver oncogenic alterations. Antibody–cytotoxic drug conjugates including trastuzumab deruxtecan have shown an excellent efficacy for NSCLC with HER2 mutations. We have now performed a phase II study to evaluate the efficacy of ado-trastuzumab emtansine (T-DM1) for NSCLC positive for HER2 exon-20 insertion mutations. Eligible patients with HER2 exon-20 insertion mutations confirmed by next-generation sequencing or multiplex polymerase chain reaction platforms and a history of one or two lines of chemotherapy received T-DM1 (3.6 mg/kg) intravenously every 21 days. The primary end-point of the study was the objective response rate (ORR). Between February 2019 and July 2020, 22 patients were enrolled in the study. A775_G776insYVMA was the most frequent HER2 exon-20 insertion mutation, accounting for 19 (86.4%) of the 22 patients. The ORR was 38.1% (90% confidence interval, 23.0–55.9%), and the disease control rate was 52.4%. The median duration of response was 3.5 months, and the median progression-free survival and median overall survival were 2.8 and 8.1 months, respectively. Toxicity was mild, with the frequency of adverse events of grade ≥3 being low. T-DM1 is a potential treatment option for patients with NSCLC with HER2 exon-20 insertion mutations. Further investigation of biomarkers for T-DM1 is warranted to improve its efficacy for NSCLC with such mutations. JapicCTI-194620 • Ado-trastuzumab emtansine was tested for patients with non-small cell lung cancer. • The objective response rate for T-DM1 in these patients was 38.1%. • Toxicity was mild, with the frequency of adverse events of grade ≥3 being low. [ABSTRACT FROM AUTHOR]

• In situ proximity ligation assay was applied to detect Her1 homo- and heterodimers. • Dimers were detected in non–small cell lung cancer (NSCLC) cell lines and tumors. • EGFR inhibitor treatment depleted homodimers more than heterodimers in NSCLC cells. • NSCLC patients with a high hetero-/homodimer ratio had a poor outcome on osimertinib. The epidermal growth factor receptor (EGFR, also known as Her1) is a member of the human epidermal growth factor receptor (HER) family of proteins and a target of tyrosine kinase inhibitors (TKIs) in the treatment of non–small cell lung cancer (NSCLC) positive for activating mutations of EGFR. Signal transduction by HER family proteins is dependent on their homo- or heterodimerization, but little is known of the relation between the relative proportions of such dimers of Her1 and sensitivity to EGFR-TKIs. We here investigated the feasibility of assessing this relation with the in situ proximity ligation assay (PLA) technique, which is able to detect the interaction of two proteins of interest when they are in close proximity. In situ PLA was applied to detect Her1 homodimers and Her1 heterodimers in NSCLC cell lines and tissue specimens positive for EGFR activating mutations. In situ PLA allowed visualization and quantitative assessment of Her1 homodimers as well as of Her1 heterodimers with Her2, Her3, or Her4 not only in NSCLC cell lines but also in NSCLC tissue specimens obtained from various anatomic sites and by different collection methods. Treatment of NSCLC cell lines with EGFR-TKIs resulted in a decrease in the number of Her1 dimers, with the effect on homodimers being greater than that on heterodimers. A high ratio of Her1 heterodimers to homodimers was associated with poor progression-free survival in NSCLC patients treated with osimertinib. In situ PLA allows the detection of HER family dimers in NSCLC tissue, and quantitative assessment of Her1 homo- and heterodimers may prove informative for prediction of the response of NSCLC patients to EGFR-TKI treatment. [ABSTRACT FROM AUTHOR]

• Cytotoxic chemotherapy and EGFR-TKI osimertinib induced prominent cell surface expression of CRT (ecto-CRT) in six NSCLC cell lines. • Systemic anticancer therapies-triggered ecto-CRT expression in NSCLC cells was positively correlated with extent of apoptosis. • Increased plasma levels of soluble CRT in advanced NSCLC patients treated with systemic anticancer therapies may serve as an indicator of ICD. Synergistic anticancer efficacy of combination treatment with immune checkpoint inhibitors (ICIs) and platinum-based chemotherapy in patients with advanced non–small cell lung cancer (NSCLC) may be attributable in part to the phenomenon of immunogenic cell death (ICD), which is characterized by the release of damage-associated molecular patterns (DAMPs) from dying tumor cells. The ability of cytotoxic chemotherapeutic agents and molecularly targeted drugs such as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) to induce DAMPs during the treatment of NSCLC has remained unclear, however. We investigated the ability of seven cytotoxic chemotherapeutic agents and the third-generation EGFR-TKI osimertinib to induce translocation of the DAMP calreticulin to the cell surface in multiple NSCLC cell lines. The plasma concentration of soluble CRT in advanced NSCLC patients treated with cytotoxic chemotherapy or osimertinib was measured. Antimetabolites and microtubule inhibitors induced expression of CRT at the cell surface (ecto-CRT) to a greater extent than did platinum agents in six NSCLC cell lines, exhibiting higher up-regulation of phosphorylation of eukaryotic initiation factor-2α (eIF2α). Ecto-CRT expression was positively correlated with apoptosis induction in NSCLC cells treated with these various chemotherapeutic agents. The drug-induced up-regulation of ecto-CRT in NSCLC cells was attenuated by the pan-caspase inhibitor Z-VAD-FMK. Osimertinib similarly increased ecto-CRT expression in association with apoptosis induction in five EGFR -mutated NSCLC cell lines. Furthermore, the plasma concentration of soluble CRT in 16 NSCLC patients treated with single-agent pemetrexed or docetaxel and in nine EGFR -mutated NSCLC patients treated with osimertinib was increased after treatment onset. Our findings indicate that antimetabolites, microtubule inhibitors, and osimertinib are effective inducers both of CRT exposure in NSCLC cell lines and of soluble CRT release in patients with advanced NSCLC, suggesting that these agents might prove effective for promotion of antitumor immunity in combination immunotherapy. [ABSTRACT FROM AUTHOR]

Objectives The regulation of redox balance in cancer cells is an important factor in tumor development and chemoresistance, with oncogene activation having been shown to induce the generation of reactive oxygen species (ROS). Activating mutations of the epidermal growth factor receptor gene ( EGFR ) are oncogenic drivers in non–small cell lung cancer (NSCLC), but it has remained unknown whether ligand-independent EGFR signaling conferred by EGFR mutation triggers ROS generation in NSCLC cells. Materials and Methods HEK293T cells were transfected with an expression vector for mutant EGFR. The expression of CD44 variant (CD44v) isoforms in NSCLC cell lines was evaluated by flow cytometry. Cells were depleted of CD44v by RNA interference and assayed for ROS and glutathione (GSH) levels. The effect of CD44v on cisplatin sensitivity was evaluated in vitro with the MTS assay. Results EGFR signaling due to EGFR mutation increased ROS levels in transfected HEK293T cells. The expression of CD44v isoforms was found to be inversely correlated with basal ROS levels in EGFR mutation–positive NSCLC cell lines. Knockdown of CD44v induced depletion of intracellular GSH and increased ROS levels in EGFR- mutated NSCLC cells that express CD44v at a high level (CD44v high ). In addition, depletion of GSH by treatment with buthionine-[ S, R ]-sulfoximine induced marked accumulation of ROS and enhanced the cytotoxicity of cisplatin in CD44v high EGFR- mutated NSCLC cells but not in corresponding CD44v low cells. This enhancement of cisplatin cytotoxicity by GSH depletion was prevented by treatment with the antioxidant N -acetyl- l -cysteine. Knockdown of CD44v also enhanced cisplatin cytotoxicity in CD44v high EGFR mutation–positive NSCLC cells but not in CD44v low cells. Conclusion Our results thus implicate CD44v in redox adaptation and as a potential target for treatment in CD44v high EGFR- mutated NSCLC cells. [ABSTRACT FROM AUTHOR]

Objectives The combination use of gefitinib and epidermal growth factor receptor (EGFR) testing is a standard first-line therapy for patients with non-small cell lung cancer (NSCLC). Here, we examined the cost-effectiveness of this approach in Japan. Materials and methods Our analysis compared the ‘EGFR testing strategy’, in which EGFR mutation testing was performed before treatment and patients with EGFR mutations received gefitinib while those without mutations received standard chemotherapy, to the ‘no-testing strategy,’ in which genetic testing was not conducted and all patients were treated with standard chemotherapy. A three-state Markov model was constructed to predict expected costs and outcomes for each strategy. We included only direct medical costs from the healthcare payer's perspective. Outcomes in the model were based on those reported in the Iressa Pan-Asia Study (IPASS). The incremental cost-effectiveness ratio (ICER) was calculated using quality-adjusted life-years (QALYs) gained. Sensitivity and scenario analyses were conducted. Results The incremental cost and effectiveness per patient of the ‘EGFR testing strategy’ compared to the ‘no-testing strategy’ was estimated to be approximately JP¥122,000 (US$1180; US$1 = JP¥104 as of February 2014) and 0.036 QALYs. The ICER was then calculated to be around JP¥3.38 million (US$32,500) per QALY gained. These results suggest that the ‘EGFR testing strategy’ is cost-effective compared with the ‘no-testing strategy’ when JP¥5.0 million to 6.0 million per QALY gained is considered an acceptable threshold. These results were supported by the sensitivity and scenario analyses. Conclusion The combination use of gefitinib and EGFR testing can be considered a cost-effective first-line therapy compared to chemotherapy such as carboplatin–paclitaxel for the treatment for NSCLC in Japan. [ABSTRACT FROM AUTHOR]

Nearly one half of all cases of acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) for non–small-cell lung cancer (NSCLC) are due to the T790M mutation in EGFR exon 20. The T790M mutation is a C→T transition mutation at a CpG dinucleotide. DNA methylation of cytosine (5-methylcytosine (5-mC)) in CpG dinucleotides is a common DNA modification; CpG dinucleotides are considered to be mutational hotspots that cause genetic diseases and cancers through spontaneous deamination of 5-mC, resulting in C→T transition mutations. This study aimed to examine the methylation level of cytosine of EGFR codon 790 and investigate whether DNA methylation was involved in acquiring the T790M mutation. We examined 18 NSCLC tumor tissues, 7 normal lymph node tissues, and 4 NSCLC cell lines (PC9, HCC827, 11-18, and A549). 5-mC was checked by bisulfite sequencing and quantified by pyrosequencing. We found that all tissue samples and cell lines had 5-mC in EGFR codon 790. The 5-mC range was 58.4–90.8%. Our results imply that hypermethylation of the CpG dinucleotide in EGFR codon 790 leads to the C→T transition mutation, causing resistance to EGFR-TKI treatment. [ABSTRACT FROM AUTHOR]