Osimertinib and Bevacizumab Cotreatment for Untreated EGFR-Mutated NSCLC With Malignant Pleural or Pericardial Effusion (SPIRAL II): A Single-Arm, Open-Label, Phase 2 Clinical Trial.

Introduction: First-line treatment of EGFR-mutated NSCLC with erlotinib plus antiangiogenic inhibitor exhibits promising results. However, the efficacy of this combination has not been fully investigated. Therefore, we evaluated the efficacy and safety of osimertinib plus bevacizumab in patients with EGFR-mutated NSCLC complicated with malignant pleural or pericardial effusion (MPE) for whom combination therapy may be particularly effective. Methods: This single-arm, open-label, phase 2 study aimed to investigate the clinical benefits of the bevacizumab (15 mg/kg) and osimertinib (80 mg) combination in the first-line setting for advanced EGFR-mutated NSCLC with MPE. The primary end point of this study was 1-year progression-free survival (PFS). The secondary end points were objective response rate, PFS, overall survival, drainage-free survival without the need for thoracic or pericardial drainage, and safety. Results: Between January 2019 and August 2020, a total of 31 patients with EGFR-mutated NSCLC were enrolled from Japan in the study. The median PFS was 8.5 months (95% confidence interval [CI]: 5.3-11.3), the 1-year PFS was 32.1% (80% CI: 21.4-43.3), and the objective response rate was 74.2% (95% CI: 56.8-86.3). The median overall survival was not reached. The median drainage-free survival was 18.4 months (95% CI: 10.3-not estimable). Anorexia was the most common grade 3 or higher adverse event (four patients, 12.9%), followed by fatigue and dyspnea (three patients, 9.7%). No treatment-related deaths were recorded. Conclusions: Osimertinib and bevacizumab combination in patients with advanced EGFR-mutated NSCLC with MPE were safe but did not effectively increase PFS when compared with the inferred value from previous literature.

Lack of dose dependency for radiation pneumonitis after chemoradiotherapy with the use of tomotherapy for lung cancer.

A 71-year-old man with stage IIB (Union for International Cancer Control, 8th edition) non-small cell lung cancer underwent intensity-modulated radiation therapy with a dose of 66 Gy administered in 33 fractions concomitant with carboplatin and paclitaxel therapy. On computed tomography after completion of radiation therapy, ground-glass opacity, which was larger on the contralateral side, was observed, but it was not observed in the high-dose area on the ipsilateral side. Although the adverse event theoretically shows dose dependency, it was finally diagnosed as radiation pneumonitis. The presence of an atypical distribution of radiation pneumonitis should be recognized to improve the diagnosis, and it is suggested that the relative volume of the normal contralateral lung receiving a dose of ≥5 Gy is a possible risk factor for radiation pneumonitis.

Designing antioxidant peptides based on the antioxidant properties of the amino acid side-chains.

Amino acids exert characteristic antioxidant activities depending on the properties of their side residues. The hydrophobic residues were effective against peroxyl radical, while acidic residues and their analogs were effective against peroxynitrite. Peptides containing tyrosine showed different activities against different reactive oxygen species (ROS) and/or reactive nitrogen species (RNS). The number and position of tyrosine did not affect the antioxidant activity against hypochlorite ion. Against the peroxyl radical, the number of tyrosine residues affected the antioxidant activity, while its position did not have a significant effect. The tyrosine position was an important factor for the antioxidant activity against peroxynitrite. The peptide GWWW showed higher antioxidant activity against peroxyl radical than tryptophan at concentrations below 25 µM, and high activity against peroxynitrite at 250 µM. Our results suggest that antioxidant peptides against a specific target ROS or RNS can be designed based on the characteristics of the amino acid side chains.

Antioxidant activities of a peptide derived from chicken dark meat.

Antioxidant activities against hypochlorite ions and peroxyl radicals of a chicken dark meat hydrolysate digested with pepsin were examined with the myoglobin method based on the structure change of myoglobin due to redox reaction with reactive oxygen species (ROS). A peptide that showed strong antioxidant activity against the peroxyl radical was isolated from the hydrolysate using HPLC equipped with a hydrophobic-interacting column. The sequence of the first five amino acid residues of the peptide was determined as YASGR (Tyr-Ala-Ser-Gly-Arg), and this sequence matched with the amino acid residues 143-147 of chicken ß-actin (GenBank: CAA25004.1). The synthetic peptide YASGR showed very high antioxidant activity against the peroxyl radical. Antioxidant activities of the free amino acids, confirmed that the tyrosine residue of this peptide was possibly responsible for antioxidant activity.

Tolerability of nintedanib (BIBF 1120) in combination with docetaxel: a phase 1 study in Japanese patients with previously treated non-small-cell lung cancer.

BACKGROUND: This phase I, open-label study evaluated the safety/tolerability and maximum tolerated dose of second-line nintedanib combined with docetaxel in Japanese patients with advanced non-small-cell lung cancer. METHODS: Eligible patients received docetaxel 60 or 75 mg/m(2) (day 1) plus nintedanib 100, 150, or 200 mg twice daily (bid; days 2-21) in 21-day cycles. Standard 3 + 3 dose escalations were performed separately in patient cohorts with a body surface area (BSA) of less than 1.5 m(2) (BSA <1.5) and BSA greater than or equal to 1.5, respectively. RESULTS: Forty-two patients (17 BSA <1.5, 25 BSA ≥ 1.5) were treated. The maximum tolerated dose of nintedanib was 150 and 200 mg bid in patients with BSA less than 1.5 and BSA greater than or equal to 1.5 (BSA ≥ 1.5), respectively, in combination with 75 mg/m(2) of docetaxel. Dose-limiting toxicities (all grade 3 hepatic enzyme elevations) occurred in 12 patients (six per cohort). Drug-related adverse events included neutropenia (95%), leukopenia (83%), fatigue (76%), alopecia (71%), decreased appetite (67%), and elevations in alanine aminotransferase (64%) and aspartate aminotransferase (64%). All hepatic enzyme elevations were reversible and manageable with dose reduction or discontinuation. Among 38 evaluable patients, 10 (26%) had a partial response and 18 (47%) had stable disease. CONCLUSION: Continuous treatment with second-line nintedanib combined with docetaxel was manageable and showed promising signs of efficacy in Japanese patients with advanced non-small-cell lung cancer.

A phase 1 study of linifanib in combination with carboplatin/paclitaxel as first-line treatment of Japanese patients with advanced or metastatic non-small cell lung cancer (NSCLC).

INTRODUCTION: Linifanib is a potent, orally active, and selective inhibitor of vascular endothelial growth factor and platelet-derived growth factor receptor kinase activities with clinical efficacy in non-small cell lung cancer (NSCLC). This phase 1 dose-escalation study evaluated the pharmacokinetics, safety, and efficacy of linifanib in combination with carboplatin/paclitaxel in Japanese patients with advanced NSCLC. METHODS: Carboplatin (AUC = 6 mg/mL/min) and paclitaxel (200 mg/m²) were administered on day 1 of each 21-day cycle up to a maximum of six cycles. Oral linifanib (7.5 mg) was given to six patients once daily throughout all cycles and escalated to 12.5 mg/day in a second cohort of six patients. RESULTS: Twelve patients received at least one dose of linifanib. The most common adverse events were hematologic and consistent with expected toxicities with carboplatin/paclitaxel. With 12.5 mg linifanib, grade 3/4 neutropenia, leukopenia, and thrombocytopenia occurred in 100, 83, and 83 % of patients, respectively. Dose-limiting grade 4 thrombocytopenia occurred in one patient at each dose level. Linifanib pharmacokinetics was similar to that in non-Japanese patients. At 12.5 mg, linifanib Cmax was 0.32 µg/mL and AUC24 was 4.29 µg h/mL. Linifanib Cmax occurred at 2-3 h with both doses and when given alone or in combination with carboplatin/paclitaxel. Exposure to linifanib appeared to be increased by carboplatin/paclitaxel, and exposure to paclitaxel appeared to be increased by linifanib. Partial responses were observed in nine patients. CONCLUSIONS: Linifanib added to carboplatin/paclitaxel is well tolerated in Japanese patients with advanced/metastatic NSCLC. The recommended dose of linifanib with carboplatin/paclitaxel is 12.5 mg, same as for US patients.

Phase 2 study of S-1 and carboplatin plus bevacizumab followed by maintenance S-1 and bevacizumab for chemotherapy-naive patients with advanced nonsquamous non-small cell lung cancer.

BACKGROUND: A previous phase 3 trial demonstrated noninferiority in terms of overall survival for combined S-1 (an oral fluoropyrimidine) and carboplatin compared with combined paclitaxel and carboplatin as first-line treatment for advanced non-small cell lung cancer (NSCLC). In the current study, the authors evaluated the efficacy and safety of combined S-1, carboplatin, and bevacizumab followed by maintenance with S-1 and bevacizumab in chemotherapy-naive patients with advanced nonsquamous NSCLC. METHODS: Patients received carboplatin (area under the concentration-time curve, 5 mg mL(-1) per minute) and bevacizumab (15 mg/kg) on day 1 plus oral S-1 (80 mg/m2 per day) on days 1 through 14 every 21 days for up to 6 cycles. For patients without disease progression, S-1 and bevacizumab were continued until disease progression or unacceptable toxicity developed. RESULTS: Forty-eight patients were enrolled in the phase 2 study; of these, 35 patients (72.9%) completed at least 4 cycles. Most toxicities of grade ≥3 were hematologic, and no increase in relative incidence associated with maintenance with S-1 and bevacizumab was observed. The objective response rate was 54.2% (95% confidence interval, 39.2%-68.6%), and the median progression-free survival was 6.8 months (95% confidence interval, 4.3-8.2 months). CONCLUSIONS: The regimen of combined S-1, carboplatin, and bevacizumab followed by maintenance with S-1 and bevacizumab was active and feasible as first-line treatment for advanced nonsquamous NSCLC.

Phase I and pharmacokinetic study of gefitinib and S-1 combination therapy for advanced adenocarcinoma of the lung.

BACKGROUND: A phase I dose-escalation study was performed to investigate the safety and pharmacokinetics of the combination of S-1 and gefitinib in patients with pulmonary adenocarcinoma who had failed previous chemotherapy. METHODS: Patients received gefitinib at a fixed daily oral dose of 250 mg, and S-1 was administered on days 1-14 every 21 days at doses starting at 60 mg/m(2) (level 1) and escalating to 80 mg/m(2) (level 2). The primary end point of the study was determination of the recommended dose for S-1 given in combination with a fixed dose of gefitinib. RESULTS: Twenty patients were enrolled in the study. Two of the first six patients at dose level 2 experienced a dose-limiting toxicity (elevation of alkaline phosphatase of grade 3 in one patient; elevations of aspartate and alanine aminotransferases of grade 3 in the other). The recommended dose was thus determined as level 2, and an additional 11 patients were assigned to this level. All observed adverse events were well managed. The response rate was 50 % (10 of 20 patients), and the median progression-free survival (PFS) and overall survival times were 10.5 and 21.2 months, respectively. In EGFR mutation-positive patients (n = 9), seven patients achieved an objective response and the median PFS was 12.4 months, whereas none with wild-type EGFR (n = 6) responded. No pharmacokinetic interaction between S-1 and gefitinib was detected. CONCLUSIONS: The combination of S-1 and gefitinib is well tolerated and appears to possess activity against EGFR mutation-positive NSCLC.

A phase I study of S-1 with concurrent radiotherapy in elderly patients with locally advanced non-small cell lung cancer.

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.

Evaluation of antioxidant activity of leafy vegetables and beans with myoglobin method.

KEY MESSAGE : Antioxidant activity of seven leafy vegetables and four beans against five reactive oxygen species and reactive nitrogen species was clearly characterized with a protocol using myoglobin as a reporter probe. Antioxidant activity of seven leafy vegetables and four beans against peroxyl radical, hydroxyl radical, hypochlorite ion, and peroxynitrite ion has been measured using myoglobin as a reporter probe (myoglobin method). Conventional DPPH method was also used to evaluate antioxidant activity of the samples. Difference of activity against different reactive oxygen species (ROS) and reactive nitrogen species (RNS) was characterized by plotting the data in a 5-axe cobweb chart. This plot clearly showed the characteristics of the antioxidant activity of the leafy vegetables and the beans. The samples examined in this work were categorized into four groups. (1) The samples showed high antioxidant activity against all ROS and RNS: daikon sprout, spinach, Qing-geng-cai, and onion. (2) The samples showed high antioxidant activity against peroxyl radical: red bean and soy bean. (3) The samples showed high antioxidant against hypochlorite ion: broccoli floret, cabbage, and Chinese cabbage. (4) The samples showed weak antioxidant activity against all ROS and RNS: cowpea and common beans. Our protocol is probably useful to characterize antioxidant activity of the crops of different cultivars, the crops obtained in different growing environments and growing seasons, the crops harvested at different age, and the crops stored in the different conditions, as well as the changes of activity during cooking process of the crops.

Phase II study of bi-weekly irinotecan for patients with previously treated HER2-negative metastatic breast cancer: KMBOG0610B.

BACKGROUND: A trial was conducted to evaluate the feasibility, efficacy, and safety of biweekly administration of irinotecan, a novel topoisomerase I inhibitor, for patients with metastatic breast cancer (MBC) previously treated with either anthracycline-based or taxane-based chemotherapy. METHODS: Eligible patients were HER2-negative, had a performance status of 0 to 2, and had been treated previously with either anthracyclines or taxanes for MBC. Patients received irinotecan intravenously at 150 mg/m(2) on days 1 and 15 every 4 weeks. The primary end-point was feasibility, and the treatment was considered feasible if a patient was able to receive three administrations of irinotecan within the first 8 weeks, as pre-specified in the protocol. RESULTS: Eighteen patients (median age 60 years) were enrolled. Fifteen patients received irinotecan more than 3 times within the first 8 weeks, with resulting feasibility of 83.3%. The median number of treatment cycles was 2 (range 1-16) during this period, and the relative dose intensity was 91.2%. Partial response was observed for one patient, so overall response rate was 5.6%. Nine patients (50.0%) had stable disease, and overall disease control was 50.0%. Median progression-free survival and overall survival periods were 3.2 and 9.6 months, respectively. The only grade 3/4 hematological toxicity was neutropenia (22.2%). Grade 3/4 non-hematological toxicities were anorexia (11.2%), diarrhea (11.2%), and fatigue (5.6%). No treatment-related death occurred. CONCLUSIONS: This study demonstrated that biweekly administration of 150 mg/m(2) irinotecan was feasible for patients with MBC treated previously with anthracyclines or taxanes.

Antioxidant activity of flavonoids evaluated with myoglobin method.

Antioxidant activities of four flavonoids (rutin, quercetin, luteolin, and kaempferol) and two non-flavonoids (chlorogenic acid and pyrocatechol) against four reactive oxygen species (ROS) have been measured with a myoglobin method developed by our group. The myoglobin method uses the absorbance changes of myoglobin (a probe molecule) due to the reaction with the ROS as an indicator for the antioxidant activity measurement. Myoglobin protective ratio (MPR) was defined to express the antioxidant activities of the specimens. Antioxidant activities against hypochlorite ion, hydroxyl radical, peroxyl radical, and peroxynitrite were measured with the myoglobin method. The antioxidant activities were comprehensively evaluated by plotting MPR against four ROS and vitamin C equivalent concentration evaluated by DPPH quenching method in 5-axe cobweb charts. The four flavonoids show a very similar pattern in the 5-axe cobweb charts, while the patterns of two non-flavonoids are quite different from that of the flavonoids. This procedure combining the myoglobin method with the cobweb charts is useful in the evaluation of antioxidant activities of plant-derived food, and also can be extended to monitor antioxidant condition of media for plant cell cultures.

Inhibition strength of short peptides derived from an ACE inhibitory peptide.

A series of peptides, derived from an ACE inhibitory peptide (VTVNPYKWLP) found in our previous work, were synthesized. Their half maximal inhibition concentrations (IC(50)) for ACE inhibition have been determined. The effect of amino acid sequence on ACE inhibition was discussed on the basis of IC(50) of the synthetic peptides, and the following characteristics of the ACE inhibitory peptide have been clarified. First, the active portion of this peptide for ACE inhibition is KW. Second, the amino acid sequences near this dipeptide (KW) have a strong effect on the inhibitory activity. Especially, the proline residue in the C-terminal end strongly enhanced ACE inhibition. It should be noted that the IC(50) value of KWLP (5.5 µM) is the same as the ACE inhibitory peptide (VTVNPYKWLP) and that the IC(50) value of KW is 7.8 µM. The stability and absorption efficiency in vivo would be significantly improved by shortening the peptide length from 10 amino acids to four amino acids or two amino acids.

Phase I safety, pharmacokinetic, and biomarker study of BIBF 1120, an oral triple tyrosine kinase inhibitor in patients with advanced solid tumors.

BIBF 1120 is an oral multitargeted tyrosine kinase inhibitor that blocks the activity of vascular endothelial growth factor (VEGF) and other growth factor receptors. We have done a phase I study to evaluate the safety, pharmacokinetics, and pharmacodynamic biomarkers of BIBF 1120. Patients with advanced refractory solid tumors were treated with BIBF 1120 at oral doses of 150 to 250 mg twice daily. Drug safety and pharmacokinetics were evaluated, as were baseline and post-treatment levels of circulating CD117-positive bone marrow-derived progenitor cells and plasma soluble VEGF receptor 2 as potential biomarkers for BIBF 1120. Twenty-one patients were treated at BIBF 1120 doses of 150 (n = 3), 200 (n = 12), or 250 mg twice daily (n = 6). Dose-limiting toxicities of reversible grade 3 or 4 elevations of liver enzymes occurred in 3 of 12 patients at 200 mg twice daily and 3 of 6 patients at 250 mg twice daily. Stable disease was achieved in 16 (76.2%) patients, and median progression-free survival was 113 days (95% confidence interval, 77-119 d). Pharmacokinetic analysis indicated that the maximum plasma concentration and area under the curve for BIBF 1120 increased with the dose within the dose range tested. Levels of CD117-positive bone marrow-derived progenitors and soluble VEGF receptor 2 decreased significantly during treatment over all BIBF 1120 dose cohorts. In conclusion, the maximum tolerated dose of BIBF 1120 in the current study was determined to be 200 mg twice daily, and our biomarker analysis indicated that this angiokinase inhibitor is biologically active.

Novel angiotensin-converting enzyme (ACE) inhibitory peptides derived from boneless chicken leg meat.

Four peptides that inhibit angiotensin-converting enzyme (ACE) were separated from the hydorlysate of boneless chicken leg meat digested with artificial gastric juice (pepsin). Two peptides were identified as the peptides encrypted in myosin heavy chain. The peptide P1 (MNVKHWPWMK) corresponds to the amino acid sequence from amino acids 825 to 834 of myosin heavy chain, and the peptide P4 (VTVNPYKWLP) corresponds to the amino acid sequence from amino acids 125 to 135 of myosin heavy chain. They are novel ACE inhibitory peptides derived from chicken, and IC(50) values of P1 and P4 were determined as 228 and 5.5 microM, respectively. Although these values were much larger than 0.022 microM for captopril, a typical synthetic ACE inhibitor, they are comparable to IC(50) values reported for various ACE inhibitory peptides derived from foods. Because the peptide P4 has a relatively low IC(50) value, it is a good starting substance for designing food supplements for hypertensive patients.

Phase I study of YM155, a novel survivin suppressant, in patients with advanced solid tumors.

PURPOSE: YM155, a novel molecular targeted agent, suppresses survivin, a member of the inhibitor of apoptosis protein family that is overexpressed in many tumor types. The aim of this study was to determine the maximum tolerated dose (MTD) and to assess the safety, pharmacokinetics, and antitumor activity of YM155 in patients with advanced refractory solid tumors. EXPERIMENTAL DESIGN: Patients with advanced refractory solid tumors were treated with escalating doses of YM155 administered by continuous i.v. infusion for 168 hours in 21-day cycles. RESULTS: Of the 34 patients enrolled, 33 (median age, 59 years) received at least 1 dose of YM155 (range, 1-19 cycles). The dose levels studied were 1.8, 3.6, 4.8, 6.0, 8.0, and 10.6 mg/m(2)/d. The MTD was determined to be 8.0 mg/m(2)/d, based on a dose-limiting toxicity of increased blood creatinine observed in 2 patients receiving 10.6 mg/m(2)/d. The most common adverse reactions judged to be related to YM155 were urine microalbumin present; fever; injection-site phlebitis; fatigue; and decreased hemoglobin/anemia, blood albumin, and lymphocyte count. The pharmacokinetic profile was almost linear over the dosing range and was similar between cycles 1 and 2. Urinary excretion of YM155 showed no definite difference among doses. Stable disease was achieved in nine patients. CONCLUSIONS: YM155 was safely administered to patients with advanced refractory solid tumors by 168-hour continuous i.v. infusion in 21-day cycles. The MTD was determined to be 8.0 mg/m(2)/d. The safety profile, plasma concentrations achieved, and antitumor activity observed merit further studies with this survivin suppressant, alone and in combination regimens.

Tandem multimer expression of angiotensin I-converting enzyme inhibitory peptide in Escherichia coli.

It is common for small tandem peptide multimer genes to be indirectly inserted into expression vectors and fused with a protein tag. In this study, a multimer of the tandem angiotensin I-converting enzyme inhibitory peptide (ACE-IP) gene was directly transferred to a commercially available vector and the designed gene was expressed as a repeated peptide in Escherichia coli BL21(DE3)pLysS. The process further developed in our study was the construction of six-repeated ACE-IP synthetic genes and their direct insertion. Protein expression in inclusion bodies was confirmed by SDS-PAGE and Western blot. Acid hydrolysis of inclusion bodies produced single-unit peptides through cleavage of the aspartyl-prolyl bonds. This cleaved recombinant peptide (rACE-IP) was purified using immuno-affinity chromatography followed by reversed phase-HPLC. 105-115 mg of the lyophilized recombinant peptide was obtained from 1 L E. coli culture. In vitro biological activity of rACE-IP was indistinguishable from that of the natural peptide produced by hydrolysis in artificial gastric juice or by acidic hydrolysis. The rACE-IP prepared by recombinant DNA technology and solid-phase synthesis methods showed a similar IC(50). This strategy could be used for the expression of important peptides, which have N-terminal proline (P) and C-terminal aspartic acid residues (D) for commercial applications, e.g. functional foods and drinks.

Screening of ACE-inhibitory peptides from a random peptide-displayed phage library using ACE-coupled liposomes.

Angiotensin I converting enzyme (ACE)-inhibitory peptides were screened from a random peptide-displayed phage library using ACE-coupled liposomes. Among four kinds of inhibitory peptides selected by biopanning with two different elution strategies, a peptide (LSTLRSFCA) showed the highest inhibitory activity with an IC(50) value of 3microM. By measuring inhibitory activities of fragments of the peptide, it was found that the RSFCA region was a functional site to inhibit strongly the ACE catalytic activity, and particularly both Arg and Cys residues were essential for the strong inhibitory activity. The inhibitory activity of RRFCA was slightly increased, while that of the RSFRA, in which the Cys residue was replaced by Arg, was decreased to greater extent in comparison with the inhibitory activity of RSFCA. Taking into account the results obtained from the SPOT analysis, it was suggested that the Arg and Phe residues in RSFCA were important for a specific interaction with ACE, and the Cys residue inhibited the ACE activity. The cystein-based ACE-inhibitory peptides have not been isolated from processed food materials. These findings suggested that the biopanning method utilizing protein-coupled liposomes and random peptide libraries might have a possibility to screen new functional peptides that are not found in processed food materials.

Differential constitutive activation of the epidermal growth factor receptor in non-small cell lung cancer cells bearing EGFR gene mutation and amplification.

The identification of somatic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) in patients with non-small cell lung cancer (NSCLC) and the association of such mutations with the clinical response to EGFR tyrosine kinase inhibitors (TKI), such as gefitinib and erlotinib, have had a substantial effect on the treatment of this disease. EGFR gene amplification has also been associated with an increased therapeutic response to EGFR-TKIs. The effects of these two types of EGFR alteration on EGFR function have remained unclear, however. We have now examined 16 NSCLC cell lines, including eight newly established lines from Japanese NSCLC patients, for the presence of EGFR mutations and amplification. Four of the six cell lines that harbor EGFR mutations were found to be positive for EGFR amplification, whereas none of the 10 cell lines negative for EGFR mutation manifested EGFR amplification, suggesting that these two types of EGFR alteration are closely associated. Endogenous EGFRs expressed in NSCLC cell lines positive for both EGFR mutation and amplification were found to be constitutively activated as a result of ligand-independent dimerization. Furthermore, the patterns of both EGFR amplification and EGFR autophosphorylation were shown to differ between cell lines harboring the two most common types of EGFR mutation (exon 19 deletion and L858R point mutation in exon 21). These results reveal distinct biochemical properties of endogenous mutant forms of EGFR expressed in NSCLC cell lines and may have implications for treatment of this condition.