Genomic origin and EGFR-TKI treatments of pulmonary adenosquamous carcinoma.

BACKGROUND: Adenosquamous carcinoma (ASC) of the lung is a heterogeneous disease that is composed of both adenocarcinoma components (ACC) and squamous cell carcinoma components (SCCC). Their genomic profile, genetic origin, and clinical management remain controversial. PATIENTS AND METHODS: Resected ASC and metastatic tumor in regional lymph nodes (LNs) were collected. The ACC and SCCC were separated by microdissection of primary tumor. The 1021 cancer-related genes were evaluated by next-generation sequencing independently in ACC and SCCC and LNs. Shared and private alterations in the two components were investigated. In addition, genomic profiles of independent cohorts of adenocarcinomas and squamous cell carcinomas were examined for comparison. We have also carried out a retrospective study of ASCs with known EGFR mutation status from 11 hospitals in China for their clinical outcomes. RESULTS: The most frequent alterations in 28 surgically resected ASCs include EGFR (79%), TP53 (68%), MAP3K1 (14%) mutations, EGFR amplifications (32%), and MDM2 amplifications (18%). Twenty-seven patients (96%) had shared variations between ACC and SCCC, and pure SCCC metastases were not found in metastatic LNs among these patients. Only one patient with geographically separated ACC and SCCC had no shared mutations. Inter-component heterogeneity was a common genetic event of ACC and SCCC. The genomic profile of ASC was similar to that of 170 adenocarcinomas, but different from that of 62 squamous cell carcinomas. The incidence of EGFR mutations in the retrospective analysis of 517 ASCs was 51.8%. Among the 129 EGFR-positive patients who received EGFR-TKIs, the objective response rate was 56.6% and the median progression-free survival was 10.1 months (95% confidence interval: 9.0-11.2). CONCLUSIONS: The ACC and SCCC share a monoclonal origin, a majority with genetically inter-component heterogeneity. ASC may represent a subtype of adenocarcinoma with EGFR mutation being the most common genomic anomaly and sharing similar efficacy to EGFR TKI.

[Chinses expert consensus on the multidisciplinary team diagnosis and treatment of lung cancer].

Lung cancer is by far the most common cancer and the leading cause of cancer death in China. Through multidimensional discussion and analysis of disease, the multidisciplinary team (MDT) diagnosis and treatment brings lots of benefits for cancer patients, including increasing patient satisfaction, reducing hospitalization expense, shortening treatment waiting time, providing more reasonable diagnosis and treatment pathways and strategies, relieving medical disputes, increasing enrollment opportunities for patients in high-quality clinical trials, patients'prognosis and life quality and so on. Presently, lung cancer MDT in China needs to be improved, including guideline following, democratic decision, landing performance and feedback, meeting records, patient follow-up and so on. So this consensus combines lung cancer MDT experience of China with leading-edge global oncology MDT experience to construct patient-centered lung cancer MDT diagnosis and treatment model, including MDT responsibility and obligations, organizational framework, working modality, standard procedures, assessment methods, and encouragement mechanisms and so on. Chinese Thoracic Oncology Group; Chinese Society of Lung Cancer; Lung Cancer Group of Oncology Branch, Chinese Medical Association; Multidisciplinary Team Diagnosis and Treatment Committee, Chinses Medical Doctor Association jointly publish this consensus. The purpose of this consensus is to provide procedures and criteria for lung cancer MDT of China.

Unique genetic profiles from cerebrospinal fluid cell-free DNA in leptomeningeal metastases of EGFR-mutant non-small-cell lung cancer: a new medium of liquid biopsy.

Background: Leptomeningeal metastases (LM) are more frequent in non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations. Due to limited access to leptomeningeal lesions, the purpose of this study was to explore the potential role of cerebrospinal fluid (CSF) as a source of liquid biopsy in patients with LM. Patients and methods: Primary tumor, CSF, and plasma in NSCLC with LM were tested by next-generation sequencing. In total, 45 patients with suspected LM underwent lumbar puncture, and those with EGFR mutations diagnosed with LM were enrolled. Results: A total of 28 patients were enrolled in this cohort; CSF and plasma were available in 26 patients, respectively. Driver genes were detected in 100% (26/26), 84.6% (22/26), and 73.1% (19/26) of samples comprising CSF cell-free DNA (cfDNA), CSF precipitates, and plasma, respectively; 92.3% (24/26) of patients had much higher allele fractions in CSF cfDNA than the other two media. Unique genetic profiles were captured in CSF cfDNA compared with those in plasma and primary tissue. Multiple copy number variations (CNVs) were mainly identified in CSF cfDNA, and MET copy number gain identified in 47.8% (11/23) of patients was the most frequent one, while other CNVs included ERBB2, KRAS, ALK, and MYC. Moreover, loss of heterozygosity (LOH) of TP53 was identified in 73.1% (19/26) CSF cfDNA, which was much higher than that in plasma (2/26, 7.7%; P < 0.001). There was a trend towards a higher frequency of concomitant resistance mutations in patients with TP53 LOH than those without (70.6% versus 33.3%; P = 0.162). EGFR T790M was identified in CSF cfDNA of 30.4% (7/23) of patients who experienced TKI progression. Conclusion: CSF cfDNA could reveal the unique genetic profiles of LM and should be considered as the most representative liquid biopsy medium for LM in EGFR-mutant NSCLC.

A phase III randomised controlled trial of erlotinib vs gefitinib in advanced non-small cell lung cancer with EGFR mutations.

BACKGROUND: A phase III trial was conducted to compare the safety and efficacy of erlotinib with that of gefitinib in advanced non-small cell lung cancer harbouring epidermal growth factor receptor mutations in exon 19 or 21. METHODS: Eligible patients were randomised to receive erlotinib (150 mg per day) or gefitinib (250 mg per day) orally until disease progression or unacceptable toxicity. We aimed to determine whether erlotinib is superior to gefitinib in efficacy. The primary end point was progression-free survival. RESULTS: A total of 256 patients were randomised to receive erlotinib (N=128) or gefitinib (N=128). Median progression-free survival was not better with erlotinib than with gefitinib (13.0 vs 10.4 months, 95% confidence interval (CI) 0.62-1.05, P=0.108). The corresponding response rates and median overall survival were 56.3% vs 52.3% (P=0.530) and 22.9 vs 20.1 months (95% CI 0.63-1.13, P=0.250), respectively. There were no significant differences in grade 3/4 toxicities between the two arms (P=0.172). CONCLUSIONS: The primary end point was not met. Erlotinib was not significantly superior to gefitinib in terms of efficacy in advanced non-small cell lung cancer with epidermal growth factor receptor mutations in exon 19 or 21, and the two treatments had similar toxicities.

Serum C-reactive protein predicts poor prognosis in patients with locoregionally advanced nasopharyngeal carcinoma treated with chemoradiotherapy.

BACKGROUND: We aimed to evaluate the association of serum C-reactive protein (crp) with prognosis in patients with locoregionally advanced nasopharyngeal carcinoma treated with chemoradiotherapy. METHODS: We retrospectively reviewed 79 patients with locoregionally advanced nasopharyngeal carcinoma (cT3-4N0-3M0) treated with chemoradiotherapy. Chemoradiotherapy consisted of external-beam radiotherapy to the nasopharynx (70-80 Gy), the lymph node-positive area (60-70 Gy), and the lymph node-negative area (50-60 Gy) combined with 3 cycles of various platinum-based regimens delivered at 3-week intervals. Elevated crp was defined as more than 8 mg/L. The survival rate was calculated using the Kaplan-Meier method, and univariate and multivariate analyses (Cox proportional hazards model) were used to identify factors significantly associated with prognosis. RESULTS: During the median follow-up of 3.9 years (range: 1-5.5 years), 23 patients died from nasopharyngeal cancer. The 5-year cancer-specific survival (css) rate was 62.90%. Before chemoradiotherapy, 18 patients had high serum crp; the css rate in that subgroup was significantly worse than the rate in the remaining patients (p = 0.0002). Multivariate analysis showed that crp was an independent prognostic indicator of css, with a hazard ratio of 3.04 (95% confidence interval: 1.22 to 7.55; p = 0.017). Among the 18 patients with elevated serum crp, 9 achieved normal serum crp after chemoradiotherapy, of whom 5 remained living with no evidence of recurrence or metastasis during follow-up. By contrast, the remaining 9 patients in whom serum crp did not normalize after chemoradiotherapy died within 4.2 years. CONCLUSIONS: Elevated serum crp before treatment predicts poor prognosis in patients with locoregionally advanced nasopharyngeal carcinoma treated with chemoradiotherapy.

Pemetrexed versus gefitinib as a second-line treatment in advanced nonsquamous nonsmall-cell lung cancer patients harboring wild-type EGFR (CTONG0806): a multicenter randomized trial.

BACKGROUND: CTONG0806 assessed the efficacy of pemetrexed versus gefitinib as second-line treatment in advanced nonsquamous nonsmall-cell lung cancer (NSCLC) harboring wild-type epidermal growth factor receptor (EGFR). PATIENTS AND METHODS: Patients with locally advanced or metastatic nonsquamous NSCLC harboring wild-type EGFR, detected by direct sequencing, and previously treated with platinum-based chemotherapy were randomized to receive gefitinib (250 mg/day) orally or pemetrexed (500 mg/m(2)) i.v. on day 1 of a 21-day cycle until disease progression or unacceptable toxicity. The primary end point was progression-free survival (PFS). The Independent Review Committee (IRC) evaluated all pictorial data. RESULTS: From February 2009 to August 2012, 161 patients were enrolled, and 157 were assessable (81 in the gefitinib arm, 76 in the pemetrexed arm). Baseline characteristics were balanced between the two arms. The median PFSs were 4.8 versus 1.6 months in the pemetrexed and gefitinib arms, respectively [hazard ratio (HR) 0.54, 95% confidence interval (CI) 0.40-0.75, P < 0.001] as confirmed by IRC evaluation (5.6versus 1.7 months, HR 0.53, 95% CI 0.38-0.75, P < 0.001). The median overall survival (OS) showed a trend of superiority in the pemetrexed arm (12.4 versus 9.6 months, HR 0.72, 95% CI 0.49-1.04, P = 0.077). Quality-of-life assessment showed no marked difference between the arms. No unexpected adverse events were found. Of 108 patients with sufficient DNA samples, EGFR mutation status was re-tested by Scorpion amplification refractory mutation system (ARMS); 32 (29.6%) tested positive (19 in the pemetrexed arm, 13 in the gefitinib arm; median PFS: 8.1 versus 7.0 months, HR 0.94, 95% CI 0.43-2.08, P = 0.877). CONCLUSIONS: CTONG0806 is the first trial to show significant improvement in PFS and an improved OS trend with pemetrexed compared with gefitinib as second-line setting treatment of EGFR wild-type advanced nonsquamous NSCLC. ARMS is superior to direct sequencing in excluding false-negative patients. CLINICALTRIALSGOV IDENTIFIER: NCT00891579.

Bevacizumab plus chemotherapy for patients with advanced pulmonary adenocarcinoma harboring EGFR mutations.

PURPOSE: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) and bevacizumab plus chemotherapy were effective for EGFR-mutant patients. However, the appropriated treatment orders remained controvertible. We investigated the efficacy of treatment orders between bevacizumab plus chemotherapy and EGFR-TKIs for EGFR-mutant patients with advanced pulmonary adenocarcinoma. PATIENTS AND METHODS: This study involved 40 EGFR-mutant patients with advanced pulmonary adenocarcinoma who were treated with bevacizumab plus carboplatin and paclitaxel (Bev + CP) and EGFR-TKIs in different treatment orders or gemcitabine plus cisplatin (GP) in first-line setting. Seventeen patients were treated with Bev + CP and 10 cases with GP in first-line treatment. Thirteen patients received EGFR-TKIs after first-line Bev + CP regimen, while 13 patients were treated with first-line EGFR-TKIs. Progression-free survival (PFS), the response rate (ORR) and overall survival (OS) were evaluated. RESULTS: Median PFS of Bev + CP treatment was significantly longer in first-line than non-first-line settings (11.7 vs. 5.6 months, P = 0.003). Median OS was 37.8 months for EGFR-mutant patients with first-line Bev + CP followed by second-line EGFR-TKIs and 31.0 months for those with first-line EGFR-TKIs and non-first-line Bev + CP, respectively (P = 0.509). Median PFS was 11.7 (95% CI 10.6-12.8) months for Bev + CP group and 4.7 (95% CI 4.4-5.0) months for GP group with the hazard ratio of 0.17 (P = 0.001). ORR was 70.6 and 50.0% in the two groups, respectively (P = 0.415). However, there was no significant difference in median OS (33.7 vs 27.8 months, P = 0.293). CONCLUSIONS: First-line Bev + CP followed by EGFR-TKIs might possibly provide favorable prognosis for EGFR-mutant patients. Bev + CP regimen significantly prolonged PFS in first-line than non-first-line settings. These findings warrant further investigations.

Toxicokinetics in drug development: an overview of toxicokinetic application in the development of PNU-101017, an anxiolytic drug candidate.

The importance of toxicokinetics in the drug development has been identified in the last decade. The main objectives of toxicokinetics in general are to define the drug bioavailability, dose proportionality, gender differences, and species differences in pharmacokinetics and metabolism, from which the target organ toxicity can be predicted and the safety doses in the first human clinical trial can be established. Toxicokinetic studies may also serve as a tool for the toxicologic pathologist in understanding models used for predicting and assessing drug-related toxic response. Toxicokinetics/toxicodynamics are critical to investigating the toxicological mechanism and understanding the comparative toxicity between animals and humans. This report presents an overview of the application of toxicokinetics and its impact in the drug development of PNU-101017, a drug candidate for the treatment of anxioety. Serial specifically designed toxicokinetic studies identified a steep dose-response relationship between the clinical signs and PNU-101017 serum or CSF concentrations, characterized the centrally mediated respiratory depression as the toxicity leading to the lethality, and demonstrated marked species differences in the sensitivity to the toxic effects. These findings lead to a termination of PNU-101017 development due to the safety concern in humans.

Piperazine imidazo[1,5-a]quinoxaline ureas as high-affinity GABAA ligands of dual functionality.

A series of imidazo[1,5-a]quinoxaline piperazine ureas appended with a tert-butyl ester side chain at the 3-position was developed. Analogues within this series have high affinity for the gamma-aminobutyric acid A (GABAA)/benzodiazepine receptor complex with efficacies ranging from inverse agonists to full agonists. Many analogues were found to be partial agonists as indicated by [35S]TBPS and Cl- current ratios. Uniquely, a number of these analogues were found to have a bell-shaped dose-response profile in the alpha1 beta2 gamma2 subtype as determined by whole cell patch-clamp technique, where in vitro efficacy was found to decrease with increasing drug concentration. Many of the compounds from this series were effective in antagonizing metrazole-induced seizures, consistent with anticonvulsant and possibly anxiolytic activity. Additionally, several analogues were also effective in lowering cGMP levels (to control values) after applied stress, also consistent with anxiolytic-like properties. The most effective compounds in these screens were also active in animal models of anxiety such as the Vogel and Geller assays. The use of the piperazine substituent allowed for excellent drug levels and a long duration of action in the central nervous system for many of the quinoxalines, as determined by ex vivo assay. Pharmacokinetic analysis of several compounds indicated excellent oral bioavailability and a reasonable half-life in rats. From this series emerged two partial agonists (55, 91) which had good activity in anxiolytic models, acceptable pharmacokinetics, and minimal benzodiazepine-type side effects.

Enantiospecific determination of PNU-83894 and its major metabolite, PNU-83892, in plasma, and its application to the characterization of the enantioselective pharmacokinetics of PNU-83894 in the dog.

A chiral method for the simultaneous analysis of the (+)- and (-)-enantiomers of PNU-83894 and its metabolite, PNU-83892, in plasma was developed to characterize the enantioselective pharmacokinetics of PNU-83894, a potential anticonvulsant candidate. The method involves solid-phase extraction (phenyl column) of the enantiomers from plasma followed by direct enantioselective separation on a beta-cyclodextrin HPLC chiral column and UV detection at 230 nm. The linear range for this method was found to be 12.5 ng/ml to 5.00 microg/ml and the intra- and inter-assay precision and accuracy for each enantiomer were <11% in all cases. The validity of this assay was also demonstrated by its application to the pharmacokinetic evaluation of PNU-83894 in the dog.

Quantitative determination of cis-3,4-dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzamide and three of its metabolites in plasma by high-performance liquid chromatography.

A sensitive HPLC method was developed for simultaneous quantitation of cis-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzamide and three of its metabolites in dog plasma. The method involved selective solid-phase extraction of the compounds of interest from dog plasma and HPLC separation of the analytes on a cyano column. Absorbance of the column effluent was monitored at 230 nm by a UV detector. The analytical procedure has a linear range of 10 ng/mL to 20 micrograms/mL, with a low limit of quantitation of 10 ng/mL for each analyte. The accuracy and intra- and interassay precision for each compound were < or = 11% in the concentration range evaluated. Applicability of this method to the quantitation of 1 and its metabolites was assessed in a preclinical pharmacokinetic study.

Application of solid-phase extraction in the method development for determination of SEPA, an acronym for soft enhancement of percutaneous absorption, in human, rat, and rabbit serum using GC-FID method.

A new nonaqueous topical minoxidil formulation containing SEPA (2-n-nonyl-1,3-dioxolane) for enhancement of percutaneous absorption was under evaluation. SEPA does not have chromophore for either ultraviolet or fluorescence detection using liquid chromatography and has no functional groups for derivatization. Therefore, a direct gas-chromatographic method with flame-ionization detection (GC-FID) was developed. Owing to the limited detection response of the FID detection, it needs a selective and concentrated extract for GC-FID analysis to improve the assay sensitivity to meet the requirement for pharmacokinetic evaluation after topical application. In addition, SEPA is a very volatile compound. Any extraction procedures involving evaporation will result in a poor recovery. The application of solid-phase extraction (SPE) makes it possible to achieve a selective and a 10-fold concentrated extract with an absolute extraction recovery of approximately 90%, which greatly improved the assay sensitivity. This method involved the extraction of SEPA and the internal standard (2-n-heptyl-1,3-dioxolane) from serum (0.1-1 ml) with 100 microliter of hexane-chloroform (1:1, v:v) using a 50 mg 1.0 ml-1 phenyl SPE column (Varian, Harbor City, CA, USA), followed by direct GC-FID analysis on a fused-silica column chemically bonded with cross-linked methyl silicone gum phase (Hewlett Packard Ultra-1, 12 m x 0.2 mm x 0.33 micron, Avondale, PA, USA). The assay demonstrated a lower limit of quantitation of 2.5 ng ml-1 and a linear range of 2.5 to 250 ng ml-1 with intra- and inter-assay precision and accuracy of < or = 10%.

Determination of 6-chloro-3-(3-cyclopropyl 1,2,4-oxadiazol-5-yl)-5-methyl-imidazo less than 1,5-a greater than-quinoxalin-4(5h)-one in rat serum, urine and brain by solid-phase extraction and liquid chromatography.

A simple, rapid, and accurate liquid chromatographic method with ultraviolet detection and solid-phase extraction is described for the quantitation of 6-chloro-3-(3-cyclopropyl 1,2,4-oxadiazol-5-yl)-5-methyl-imidazo less than 1,5-a greater than-quinoxalin-4(5h)-one (I, U-80447) in rat serum, urine and brain. Linear calibration curves were obtained in the concentration ranges of 5 ng ml-1-20 micrograms ml-1 (serum), 20 ng ml-1-20 micrograms ml-1 (urine), and 50 ng g-1-200 micrograms g-1 (brain). Intra- and inter-assay precision and accuracy were all found to be less than 10% at the three concentrations evaluated. The absolute extraction recovery each from serum, urine and brain was greater than or equal to 90%. Application of this method to the quantitation of the title compound in rat serum and brain for a pharmacokinetic study is reported.

Simultaneous quantitation of pioglitazone and its metabolites in human serum by liquid chromatography and solid phase extraction.

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of pioglitazone (U-72107) and its potential metabolites (M-1 to M-6) in human serum was developed. The method involved a solid phase extraction (SPE) of pioglitazone, its metabolites, and the internal standard (U-92573) from serum using C18 SPE columns with an elution solvent of 0.5 ml of acetonitrile-water (35:65, v/v). Separation of the eight analytes was achieved within 20 min using a reversed-phase Zorbax RX-C8 analytical column (250 mm x 4.6 mm i.d., 5 microns particle size) with a mobile phase of acetonitrile-water (40:60, v/v) containing 3 ml acetic acid per liter mobile phase (apparent pH 5.5). An ultraviolet detector operated at 269 nm was used with a linear response observed from 0.02 to 2 micrograms ml-1 for these analytes except for M-4 which was best fitted with a polynomial regression. Limit of quantitation was found to be 0.02 microgram ml-1 for pioglitazone, M-3, M-5, and M-6; 0.04 microgram ml-1 for M-2 and M-4; and 0.5 microgram ml-1 for M-1 when using a 0.5 ml serum sample for extraction. Obtained from the method validation, intra- and inter-assay precision was < or = 9% and accuracy ranged from -8.2 to 13.4% for all analytes. The applicability of this method has been demonstrated by successfully analyzing clinical serum samples. The strategies in the HPLC characterization and in the SPE procedure development for this method are discussed as well.

Development of a sensitive bioanalytical method for determination of PNU-83757 in rat, monkey and human plasma: from LC-UV to LC-MS/MS.

To support pre-clinical pharmacokinetic/toxicokinetic (PK/TK) evaluation, a sensitive bioanalytical method for determination of N-cyano-N'-(tert-pentyl)-N"-(3-pyridinyl) guanidine (PNU-83757), in rat and monkey plasma was required. Although the UV response of PNU-83757 was quite decent and the extracts using solid phase extraction (SPE) were very selective and concentrated, the best limit of quantitation (LOQ) achieved was 0.4 ng ml(-1) using 0.5 ml plasma for extraction and 2 ng ml(-1) using 0.1 ml plasma for extraction, which was insufficient for PK/TK evaluation at lower doses. When using liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometric detection (LC-APCI-MS/MS, positive ions) and SPE, a LOQ of 0.045 ng ml(-1) for PNU-83757 was reached. Quantitation was accomplished using the precursor --> product ion combinations of m/z 232 --> 162 for PNU-83757 and m/z 236 --> 166 for the internal standard, [2H(4)]PNU-83757, in the multiple reaction monitoring mode. This method has been successfully utilized for PK/TK evaluation in pre-clinical studies and proved to have sufficient sensitivity to determine plasma concentrations for a dose level as low as 1 microg kg(-1) day(-1) in the rat and monkey. Further improvement of this method by using electrospray mass spectrometric detection (LC-ESI-MS/MS, positive ions) and automated membrane SPE, gave an LOQ of 0.008 ng ml(-1), and allowed analysis of large numbers of samples to support clinical PK studies in microg dose levels.

Serum levels of selenium in patients with brain metastases from non-small cell lung cancer before and after radiotherapy.

PURPOSE: This study was to evaluate the influence of radiotherapy on the selenium serum levels of non-small cell cancer patients with brain metastases. PATIENTS AND METHODS: This prospective study included 95 non-small cell cancer patients with brain metastases treated by radiotherapy from December 2007 until November 2010. Plasma selenium levels were determined before and at the end of the radiotherapy. Age, body mass index (BMI), prior chemotherapy, pathological type and personal habits (smoking and alcoholism) were recorded for each patient. RESULTS: The mean age was 63 years; the mean BMI was 27.6. Seventy-six patients (80%) were non-smokers. Sixty-two patients (65.3%) showed no drinking habits and 8 (8.4%) have no prior chemotherapy. Thirty-nine patients (41.1%) were adenocarcinoma, 51 (53.7%) were squamous cell carcinoma and five (5.3%) were large cell carcinoma. At the beginning of radiotherapy, the mean selenium level for all patients was 90.4 µg/l and after radiation this value dropped to 56.3 µg/l. Multivariate analysis showed statistically significant difference in the plasma selenium concentration before and after radiotherapy for age (P<0.001), BMI (P<0.001), smoking (P<0.001), alcoholism (P<0.001), prior chemotherapy (P<0.001) and pathological type (P<0.001). CONCLUSION: Significant reduction in plasma levels of selenium was recorded in patients undergoing radiotherapy, suggesting attention to the nutritional status of this micronutrient and other antioxidant agents.

High-level expression of Rad51 is an independent prognostic marker of survival in non-small-cell lung cancer patients.

High-level expression of Rad51, a key factor in homologous recombination, has been observed in a variety of human malignancies. This study was aimed to evaluate Rad51 expression to serve as prognostic marker in non-small-cell lung cancer (NSCLC). A total of 383 non-small-cell lung tumours were analysed immunohistochemically on NSCLC tissue microarrays. High-level Rad51 expression was observed in 29.4% (100 out of 340) of cases. Patients whose tumours displayed high-level Rad51 expression showed a significantly shorter median survival time of 19 vs 68 months (P<0.0001, log-rank test). Similarly T status, N status, M status, clinical stage and histological tumour grade were significant prognostic markers in univariate Cox survival analysis. Importantly, Rad51 expression (P<0.0001) together with tumour differentiation (P<0.009), clinical stage (P=0.004) and N status (P=0.0001) proved to be independent prognostic parameters in multivariate analysis. Rad51 expression predicted the outcome of squamous cell cancer as well as adenocarcinoma of the lung. Our results suggest that Rad51 expression provides additional prognostic information for surgically treated NSCLC patients. We hypothesise that the decreased survival of NSCLC patients with high-level expression of Rad51 is related to an enhanced propensity of tumour cells for survival, antiapoptosis and chemo-/radioresistance.

Determination of 3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-5-(1-methylethyl)imidazo[1, 5-a]quinoxalin-4(5H)-one in serum by high-performance liquid chromatography.

A high-performance liquid chromatographic assay with solid-phase extraction (SPE) for the rapid and sensitive quantitation of 3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-5-(1-methylethyl)imidazo[1, 5-a]quinoxalin-4(5H)-one (I, U-78875) in serum is described. The validation results indicated that the present method had excellent intra- and inter-assay precision (less than or equal to 9.5%, mean +/- S.D. = 3.9 +/- 3.0%, n = 25) and accuracy (less than or equal to 10.0%, mean +/- S.D. = 3.0 +/- 2.9%, n = 25), as well as improved sensitivity (2 ng ml, using a 100-microliters injection). Each chromatographic run is only 10 min and the organic solvent for the extraction of I and internal standard (U-82217) from serum was only 300 microliters. The application results obtained from the SPE method were in good agreement with the advanced automated sample preparation method.

Application of solid-phase extraction in the determination of U-82217 in rat serum, urine and brain.

The techniques of solid-phase extraction (SPE) were applied in the analytical method development for the determination of U-82217, 3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-5-[(4-methoxyphenyl)methyl] -imidazo[1,5-a]quinoxalin-4(5H)-one, in rat serum, urine and brain. Samples of serum, urine or brain homogenate containing U-82217 were loaded on C18 SPE columns and eluted with acetonitrile (300 microliters). The prepared samples were analyzed by reversed-phase HPLC using an ODS column with a mobile phase of acetonitrile-water (45:55, v/v) containing 0.12% of acetic acid (pH 6.0 +/- 0.1). The UV absorbance of the column effluent was monitored at a wavelength of 318 nm. The absolute extraction recovery from serum, urine and brain samples was ca. 90%. Linear calibration graphs were obtained over the ranges 5 ng/ml-20 micrograms/ml (serum), 20 ng/ml-20 micrograms/ml (urine) and 50 ng/g-200 micrograms/g (brain). The intra- and inter-assay precision and accuracy were all found to be < 13% at the concentrations evaluated. The strategy in SPE development and the application of this method to the determination of U-82217 in rat serum and brain for a pharmacokinetic study are also discussed.